Antibodies to M-CSF

ABSTRACT

The present invention relates to antibodies and antigen-binding portions thereof that specifically bind to a M-CSF, preferably human M-CSF, and that function to inhibit a M-CSF. The invention also relates to human anti-M-CSF antibodies and antigen-binding portions thereof. The invention also relates to antibodies that are chimeric, bispecific, derivatized, single chain antibodies or portions of fusion proteins. The invention also relates to isolated heavy and light chain immunoglobulins derived from human anti-M-CSF antibodies and nucleic acid molecules encoding such immunoglobulins. The present invention also relates to methods of making human anti-M-CSF antibodies, compositions comprising these antibodies and methods of using the antibodies and compositions for diagnosis and treatment. The invention also provides gene therapy methods using nucleic acid molecules encoding the heavy and/or light immunoglobulin molecules that comprise the human anti-M-CSF antibodies. The invention also relates to transgenic animals and transgenic plants comprising nucleic acid molecules of the present invention.

This application is a divisional of U.S. application Ser. No.14/923,655, filed Oct. 27, 2015, which is a divisional of U.S.application Ser. No. 13/458,820, filed Apr. 27, 2012, which is acontinuation of U.S. application Ser. No. 12/748,602, filed Mar. 29,2010, and issued as U.S. Pat. No. 8,188,249, which is a continuation ofU.S. application Ser. No. 11/894,560, filed Aug. 20, 2007 and issued asU.S. Pat. No. 7,728,113, which is a continuation of U.S. applicationSer. No. 11/375,221, filed Mar. 13, 2006 and issued as U.S. Pat. No.7,326,414, which is a continuation of U.S. application Ser. No.10/938,353, filed Sep. 9, 2004 and issued as U.S. Pat. No. 7,592,430,which claims priority from U.S. Provisional Application 60/502,163,filed Sep. 10, 2003. The entire teachings of the above-identifiedapplications are incorporated herein by reference.

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII text file, created on Jun. 5, 2017, is named000659-0052-107-SL.txt, and is 172,080 bytes in size.

BACKGROUND OF THE INVENTION

Macrophage colony stimulating factor (M-CSF) is a member of the familyof proteins referred to as colony stimulating factors (CSFs). M-CSF is asecreted or a cell surface glycoprotein comprised of two subunits thatare joined by a disulfide bond with a total molecular mass varying from40 to 90 kD ((Stanley E. R., et al., Mol. Reprod. Dev., 46:4-10 (1997)).Similar to other CSFs, M-CSF is produced by macrophages, monocytes, andhuman joint tissue cells, such as chondrocytes and synovial fibroblasts,in response to proteins such as interleukin-1 or tumor necrosisfactor-alpha. M-CSF stimulates the formation of macrophage colonies frompluripotent hematopoietic progenitor stem cells (Stanley E. R., et al.,Mol. Reprod. Dev., 46:4-10 (1997)).

M-CSF typically bind to its receptor, c-fms, in order to exert abiological effect. c-fms contains five extracellular Ig domains, onetransmembrane domain, and an intracellular domain with two kinasedomains. Upon M-CSF binding to c-fms, the receptor homo-dimerizes andinitiates a cascade of signal transduction pathways including theJAK/STAT, PI3K, and ERK pathways.

M-CSF is an important regulator of the function, activation, andsurvival of monocytes/macrophages. A number of animal models haveconfirmed the role of M-CSF in various diseases, including rheumatoidarthritis (RA) and cancer. Macrophages comprise key effector cells inRA. The degree of synovial macrophage infiltration in RA has been shownto closely correlate with the extent of underlying joint destruction.M-CSF, endogenously produced in the rheumatoid joint bymonocytes/macrophages, fibroblasts, and endothelial cells, acts on cellsof the monocyte/macrophage lineage to promote their survival anddifferentiation into bone destroying osteoclasts, and enhancepro-inflammatory cellular functions such as cytotoxicity, superoxideproduction, phagocytosis, chemotaxis and secondary cytokine production.For example, treatment with M-CSF in the rat streptococcus agalactiaesonicate-induced experimental arthritis model lead to enhanced pathology(Abd, A. H., et al., Lymphokine Cytokine Res. 10:43-50 (1991)).Similarly, subcutaneous injections of M-CSF in a murine model ofcollagen-induced arthritis (CIA), which is a model for RA, resulted in asignificant exacerbation of the RA disease symptoms (Campbell I. K., etal., J. Leuk. Biol. 68:144-150 (2000)). Furthermore, MRL/lpr mice thatare highly susceptible to RA and other autoimmune diseases have elevatedbasal M-CSF serum concentrations (Yui M. A., et al., Am. J. Pathol.139:255-261 (1991)). The requirement for endogenous M-CSF in maintainingCIA was demonstrated by a significant reduction in the severity ofestablished disease by M-CSF neutralizing mouse monoclonal antibody(Campbell I. K., et al., J. Leuk. Biol. 68:144-150 (2000)).

With respect to cancer, inhibition of colony stimulating factors byantisense oligonucleotides suppresses tumor growth in embryonic andcolon tumor xenografts in mice by decelerating macrophage-mediated ECMbreakdown (Seyedhossein, A., et al., Cancer Research, 62:5317-5324(2002)).

M-CSF binding to c-fms and its subsequent activation ofmonocyte/macrophages is important in a number of disease states. Inaddition to RA and cancer, the other examples of M-CSF-related diseasestates include osteoporosis, destructive arthritis, atherogenesis,glomerulonephritis, Kawasaki disease, and HIV-1 infection, in whichmonocytes/macrophages and related cell types play a role. For instance,osteoclasts are similar to macrophages and are regulated in part byM-CSF. Growth and differentiation signals induced by M-CSF in theinitial stages of osteoclast maturation are essential for theirsubsequent osteoclastic activity in bone.

Osteoclast mediated bone loss, in the form of both focal bone erosionsand more diffuse juxta-articular osteoporosis, is a major unsolvedproblem in RA. The consequences of this bone loss include jointdeformities, functional disability, increased risk of bone fractures andincreased mortality. M-CSF is uniquely essential for osteoclastogenesisand experimental blockade of this cytokine in animal models of arthritissuccessfully abrogates joint destruction. Similar destructive pathwaysare known to operate in other forms of destructive arthritis such aspsoriatic arthritis, and could represent venues for similarintervention.

Postmenopausal bone loss results from defective bone remodelingsecondary to an uncoupling of bone formation from exuberant osteoclastmediated bone resorption as a consequence of estrogen deficiency.In-vivo neutralization of M-CSF using a blocking antibody has been shownin mice to completely prevent the rise in osteoclast numbers, theincrease in bone resorption and the resulting bone loss induced byovariectomy.

Several lines of evidence point to a central role for M-CSF inatherogenesis, and in proliferative intimal hyperplasia after mechanicaltrauma to the arterial wall. All the major cell types in atheroscleroticlesions have been shown to express M-CSF, and this is furtherup-regulated by exposure to oxidized lipoprotein. Blockade of M-CSFsignaling with a neutralizing c-fms antibody reduces the accumulation ofmacrophage-derived foam cells in the aortic root of apolipoprotein Edeficient mice maintained on a high fat diet.

In both experimental and human glomerulonephritis, glomerular M-CSFexpression has been found to co-localize with local macrophageaccumulation, activation and proliferation and correlate with the extentof glomerular injury and proteinuria. Blockade of M-CSF signaling via anantibody directed against its receptor c-fms significantlydown-regulates local macrophage accumulation in mice during the renalinflammatory response induced by experimental unilateral uretericobstruction.

Kawasaki disease (KD) is an acute, febrile, pediatric vasculitis ofunknown cause. Its most common and serious complications involve thecoronary vasculature in the form of aneurismal dilatation. Serum M-CSFlevels are significantly elevated in acute phase Kawasaki's disease, andnormalize following treatment with intravenous immunoglobulin. Giantcell arthritis (GCA) is an inflammatory vasculopathy mainly occurring inthe elderly in which T cells and macrophages infiltrate the walls ofmedium and large arteries leading to clinical consequences that includeblindness and stroke secondary to arterial occlusion. The activeinvolvement of macrophages in GCA is evidenced by the presence ofelevated levels of macrophage derived inflammatory mediators withinvascular lesions.

M-CSF has been reported to render human monocyte derived macrophagesmore susceptible to HIV-1 infection in vitro. In a recent study, M-CSFincreased the frequency with which monocyte-derived macrophages becameinfected, the amount of HIV mRNA expressed per infected cell, and thelevel of proviral DNA expressed per infected culture.

Given the role of M-CSF in various diseases, a method for inhibitingM-CSF activity is desirable.

There is a critical need for therapeutic anti-M-CSF antibodies.

SUMMARY OF THE INVENTION

The present invention provides isolated human antibodies orantigen-binding portions thereof that specifically bind human M-CSF andacts as a M-CSF antagonist and compositions comprising said antibody orportion.

The invention also provides for compositions comprising the heavy and/orlight chain, the variable regions thereof, or antigen-binding portionsthereof an anti-M-CSF antibody, or nucleic acid molecules encoding anantibody, antibody chain or variable region thereof the inventioneffective in such treatment and a pharmaceutically acceptable carrier.In certain embodiments, the compositions may further comprise anothercomponent, such as a therapeutic agent or a diagnostic agent. Diagnosticand therapeutic methods are also provided by the invention. In certainembodiments, the compositions are used in a therapeutically effectiveamount necessary to treat or prevent a particular disease or condition.

The invention also provides methods for treating or preventing a varietyof diseases and conditions such as, but not limited to, inflammation,cancer, atherogenesis, neurological disorders and cardiac disorders withan effective amount of an anti-M-CSF antibody of the invention, orantigen binding portion thereof, nucleic acids encoding said antibody,or heavy and/or light chain, the variable regions, or antigen-bindingportions thereof.

The invention provides isolated cell lines, such as a hybridomas, thatproduce anti-M-CSF antibodies or antigen-binding portions thereof.

The invention also provides nucleic acid molecules encoding the heavyand/or light chains of anti-M-CSF antibodies, the variable regionsthereof, or the antigen-binding portions thereof.

The invention provides vectors and host cells comprising the nucleicacid molecules, as well as methods of recombinantly producing thepolypeptides encoded by the nucleic acid molecules.

Non-human transgenic animals or plants that express the heavy and/orlight chains, or antigen-binding portions thereof, of anti-M-CSFantibodies are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are graphs illustrating that the anti-M-CSF antibodiesresulted in a dose-related decrease in total monocyte counts in male andfemale monkeys over time. The monocyte counts were determined by lightscatter using an Abbott Diagnostics Inc. Cell Dyn system. Monocytecounts were monitored from 24 hours through 3 weeks after administrationof vehicle or antibody 8.10.3 at 0, 0.1, 1 or 5 mg/kg in a dose volumeof 3.79 mL/kg over an approximately 5 minute period.

FIG. 1A male monkeys.

FIG. 1B female monkeys.

FIGS. 2A and 2B are graphs illustrating that anti-M-CSF treatmentresulted in a reduction in the percentage of CD14+CD16+ monocytes, inmale and female monkeys. 0-21 days after administration of vehicle orantibody 8.10.3 at 0, 0.1, 1 or 5 mg/kg in a dose volume of 3.79 mL/kgover an approximately 5 minute period. For each monkey tested, thepercentage of monocytes within the CD14+CD16+ subset was determinedafter each blood draw, on days 1, 3, 7, 14 and 21 after 8.10.3injection.

FIG. 2A male monkeys.

FIG. 2B female monkeys.

FIG. 3 is a graph illustrating that anti-M-CSF treatment resulted in adecrease in the percentage change of total monocytes at all doses ofantibody 8.10.3F as compared to pre-test levels of monocytes.

FIG. 3 shows data collected from experiments using antibody 8.10.3F.

FIGS. 4A-4ZZ provide sequence alignments of the predicted amino acidsequences of light and heavy chain variable regions from twenty-sixanti-M-CSF antibodies compared with the germline amino acid sequences ofthe corresponding variable region genes. Differences between theantibody sequences and the germline gene sequences are indicated inbold-faced type. Dashes represent no change from germline. Theunderlined sequences in each alignment represent, from left to right,the FR1, CDR1, FR2, CDR2, FR3, CDR3 AND FR4 sequences.

FIG. 4A shows an alignment of the predicted amino acid sequence of thelight chain variable region for antibody 252 (residues 21-127 of SEQ IDNO: 4) to the germline V_(κ)O12, J_(κ)3 sequence (SEQ ID NO: 103).

FIG. 4B shows an alignment of the predicted amino acid sequence of thelight chain variable region for antibody 88 (residues 21-127 of SEQ IDNO: 8) to the germline V_(κ)O12, J_(κ)3 sequence (SEQ ID NO: 103).

FIG. 4C shows an alignment of the predicted amino acid sequence of thelight chain variable region for antibody 100 (residues 21-127 of SEQ IDNO: 12) to the germline V_(κ)L2, J_(κ)3 sequence (SEQ ID NO: 107).

FIG. 4D shows an alignment of the predicted amino acid sequence of thelight chain variable region for antibody 3.8.3 (residues 23-130 of SEQID NO: 16) to the germline V_(κ)L5, J_(κ)3 sequence (SEQ ID NO: 109).

FIG. 4E shows an alignment of the predicted amino acid sequence of thelight chain variable region for antibody 2.7.3 (residues 23-130 of SEQID NO: 20) to the germline V_(κ)L5, J_(κ)4 sequence (SEQ ID NO: 117).

FIG. 4F shows an alignment of the predicted amino acid sequence of thelight chain variable region for antibody 1.120.1 (residues 21-134 of SEQID NO: 24) to the germline V_(κ)B3, J_(κ)1 sequence (SEQ ID NO: 112).

FIG. 4G shows an alignment of the predicted amino acid sequence of theheavy chain variable region for antibody 252 (residues 20-136 of SEQ IDNO: 2) to the germline V_(H)3-11, D_(H)7-27 J_(H)6 sequence (SEQ ID NO:106).

FIG. 4H shows an alignment of the predicted amino acid sequence of theheavy chain variable region for antibody 88 (residues 20-138 of SEQ IDNO: 6) to the germline V_(H)3-7, D_(H)6-13, J_(H)4 sequence (SEQ ID NO:105).

FIG. 4I shows the alignment of the predicted amino acid sequence of theheavy chain variable region for antibody 100 (residues 20-141 of SEQ IDNO: 10) to the germline V_(H)3-23, D_(H)1-26, J_(H)4 sequence (SEQ IDNO: 104).

FIG. 4J shows an alignment of the predicted amino acid sequence of theheavy chain variable region for antibody 3.8.3 (residues 20-135 of SEQID NO: 14) to the germline V_(H)3-11, D_(H)7-27, J_(H)4 sequence (SEQ IDNO: 108).

FIG. 4K shows an alignment of the predicted amino acid sequence of theheavy chain variable region for antibody 2.7.3 (residues 20-137 of SEQID NO: 18) to the germline V_(H)3-33, D_(H)1-26, J_(H)4 sequence (SEQ IDNO: 110).

FIG. 4L shows an alignment of the predicted amino acid sequence of theheavy chain variable region for antibody 1.120.1 (residues 20-139 of SEQID NO: 22) to the germline V_(H)1-18, D_(H)4-23, J_(H)4 sequence (SEQ IDNO: 111).

FIG. 4M shows an alignment of the predicted amino acid sequence of thelight chain variable region for antibody 8.10.3 (residues 21-129 of SEQID NO: 44) to the germline V_(κ)A27, J_(κ)4 sequence (SEQ ID NO: 114).

FIG. 4N shows an alignment of the predicted amino acid sequence of theheavy chain variable region for antibody 8.10.3 (residues 20-141 of SEQID NO: 30) to the germline V_(H)3-48, D_(H)1-26, J_(H)4b sequence (SEQID NO: 113).

FIG. 4O shows an alignment of the predicted amino acid sequence of thelight chain variable region for antibody 9.14.4 (residues 23-130 of SEQID NO: 28) to the germline V_(κ)O12, J_(κ)3 sequence (SEQ ID NO: 103).

FIG. 4P shows an alignment of the predicted amino acid sequence of theheavy chain variable region for antibody 9.14.4 (residues 20-135 of SEQID NO: 38) to the germline V_(H)3-11, D_(H)7-27, J_(H)4b sequence (SEQID NO: 116).

FIG. 4Q shows an alignment of the predicted amino acid sequence of thelight chain variable region for antibody 9.7.2 (residues 23-130 of SEQID NO: 48) to the germline V_(κ)O12, J_(κ)3 sequence (SEQ ID NO: 103).

FIG. 4R shows an alignment of the predicted amino acid sequence of theheavy chain variable region for antibody 9.7.2 (residues 20-136 of SEQID NO: 46) to the germline V_(H)3-11, D_(H)6-13, J_(H)6b sequence (SEQID NO: 115).

FIG. 4S shows an alignment of the predicted amino acid sequence of thelight chain variable region for antibody 9.14.4I (residues 23-130 of SEQID NO: 28) to the germline V_(κ)O12 J_(κ)3 sequence (SEQ ID NO: 103).

FIG. 4T shows an alignment of the predicted amino acid sequence of theheavy chain variable region for antibody 9.14.4I (residues 20-135 of SEQID NO: 26) to the germline V_(H)3-11, D_(H)7-27, J_(H)4b sequence (SEQID NO: 116).

FIG. 4U shows an alignment of the predicted amino acid sequence of thelight chain variable region for antibody 8.10.3F (residues 21-129 of SEQID NO: 32) to the germline V_(κ)A27, J_(κ)4 sequence (SEQ ID NO: 114).

FIG. 4V shows an alignment of the predicted amino acid sequence of theheavy chain variable region for antibody 8.10.3F (residues 20-141 of SEQID NO: 30) to the germline V_(H)3-48, D_(H)1-26, J_(H)4b sequence (SEQID NO: 113).

FIG. 4W shows an alignment of the predicted amino acid sequence of thelight chain variable region for antibody 9.7.2IF (residues 23-130 of SEQID NO: 36) to the germline V_(κ)O12, J_(κ)3 sequence (SEQ ID NO: 103).

FIG. 4X shows an alignment of the predicted amino acid sequence of theheavy chain variable region for antibody 9.7.2IF (residues 20-136 of SEQID NO: 34) to the germline V_(H)3-11, D_(H)6-13, J_(H)6b sequence (SEQID NO: 115).

FIG. 4Y shows an alignment of the predicted amino acid sequence of thelight chain variable region for antibody 9.7.2C-Ser (residues 23-130 ofSEQ ID NO: 52) to the germline V_(κ)O12, J_(κ)3 sequence (SEQ ID NO:103).

FIG. 4Z shows an alignment of the predicted amino acid sequence of theheavy chain variable region for antibody 9.7.2C-Ser (residues 20-136 ofSEQ ID NO: 50) to the germline V_(H)3-11, D_(H)6-13, J_(H)6b sequence(SEQ ID NO: 115).

FIG. 4AA shows an alignment of the predicted amino acid sequence of thelight chain variable region for antibody 9.14.4C-Ser (residues 23-130 ofSEQ ID NO: 56) to the germline V_(κ)O12, J_(κ)3 sequence (SEQ ID NO:103).

FIG. 4BB shows an alignment of the predicted amino acid sequence of theheavy chain variable region for antibody 9.14.4C-Ser (residues 20-135 ofSEQ ID NO: 54) to the germline V_(H)3-11, D_(H)7-27, J_(H)4b sequence(SEQ ID NO: 116).

FIG. 4CC shows an alignment of the predicted amino acid sequence of thelight chain variable region for antibody 8.10.3C-Ser (residues 21-129 ofSEQ ID NO: 60) to the germline V_(κ)A27, J_(κ)4 sequence (SEQ ID NO:114).

FIG. 4DD shows an alignment of the predicted amino acid sequence of theheavy chain variable region for antibody 8.10.3C-Ser (residues 20-141 ofSEQ ID NO: 58) to the germline V_(H)3-48, D_(H)1-26, J_(H)4b sequence(SEQ ID NO: 113).

FIG. 4EE shows an alignment of the predicted amino acid sequence of thelight chain variable region for antibody 8.10.3-CG2 (residues 21-129 ofSEQ ID NO: 60) to the germline V_(κ)A27, J_(κ)4 sequence (SEQ ID NO:114).

FIG. 4FF shows an alignment of the predicted amino acid sequence of theheavy chain variable region for antibody 8.10.3-CG2 (residues 20-141 ofSEQ ID NO: 62) to the germline V_(H)3-48, D_(H)1-26, J_(H)4b sequence(SEQ ID NO: 113).

FIG. 4GG shows an alignment of the predicted amino acid sequence of thelight chain variable region for antibody 9.7.2-CG2 (residues 23-130 ofSEQ ID NO: 52) to the germline V_(κ)O12, J_(κ)3 sequence (SEQ ID NO:103).

FIG. 4HH shows an alignment of the predicted amino acid sequence of theheavy chain variable region for antibody 9.7.2-CG2 (residues 20-136 ofSEQ ID NO: 66) to the germline V_(H)3-11, D_(H)6-13, J_(H)6b sequence(SEQ ID NO: 115).

FIG. 4II shows an alignment of the predicted amino acid sequence of thelight chain variable region for antibody 9.7.2-CG4 (residues 23-130 ofSEQ ID NO: 52) to the germline V_(κ)O12, J_(κ)3 sequence (SEQ ID NO:103).

FIG. 4JJ shows an alignment of the predicted amino acid sequence of theheavy chain variable region for antibody 9.7.2-CG4 (residues 20-135 ofSEQ ID NO: 70) to the germline V_(H)3-11, D_(H)6-13, J_(H)6b sequence(SEQ ID NO: 115).

FIG. 4KK shows an alignment of the predicted amino acid sequence of thelight chain variable region for antibody 9.14.4-CG2 (residues 23-130 ofSEQ ID NO: 56) to the germline V_(κ)O12, J_(κ)3 sequence (SEQ ID NO:103).

FIG. 4LL shows an alignment of the predicted amino acid sequence of theheavy chain variable region for antibody 9.14.4-CG2 (residues 20-135 ofSEQ ID NO: 74) to the germline V_(H)3-11, D_(H)7-27, J_(H)4b sequence(SEQ ID NO: 116).

FIG. 4MM shows an alignment of the predicted amino acid sequence of thelight chain variable region for antibody 9.14.4-CG4 (residues 23-130 ofSEQ ID NO: 56) to the germline V_(κ)O12, J_(κ)3 sequence (SEQ ID NO:103).

FIG. 4NN shows an alignment of the predicted amino acid sequence of theheavy chain variable region for antibody 9.14.4-CG4 (residues 20-135 ofSEQ ID NO: 78) to the germline V_(H)3-11, D_(H)7-27, J_(H)4b sequence(SEQ ID NO: 116).

FIG. 4OO shows an alignment of the predicted amino acid sequence of thelight chain variable region for antibody 9.14.4-Ser (residues 23-130 ofSEQ ID NO: 28) to the germline V_(κ)O12, J_(κ)3 sequence (SEQ ID NO:103).

FIG. 4PP shows an alignment of the predicted amino acid sequence of theheavy chain variable region for antibody 9.14.4-Ser (residues 20-135 ofSEQ ID NO: 82) to the germline V_(H)3-11, D_(H)7-27, J_(H)4b sequence(SEQ ID NO: 116).

FIG. 4QQ shows an alignment of the predicted amino acid sequence of thelight chain variable region for antibody 9.7.2-Ser (residues 23-130 ofSEQ ID NO: 48) to the germline V_(κ)O12, J_(κ)3 sequence (SEQ ID NO:103).

FIG. 4RR shows an alignment of the predicted amino acid sequence of theheavy chain variable region for antibody 9.7.2-Ser (residues 20-136 ofSEQ ID NO: 86) to the germline V_(H)3-11, D_(H)6-13, J_(H)6b sequence(SEQ ID NO: 115).

FIG. 4SS shows an alignment of the predicted amino acid sequence of thelight chain variable region for antibody 8.10.3-Ser (residues 21-129 ofSEQ ID NO: 44) to the germline V_(κ)A27, J_(κ)4 sequence (SEQ ID NO:114).

FIG. 4TT shows an alignment of the predicted amino acid sequence of theheavy chain variable region for antibody 8.10.3-Ser (residues 20-141 ofSEQ ID NO: 90) to the germline V_(H)3-48, D_(H)1-26, J_(H)4b sequence(SEQ ID NO: 113).

FIG. 4UU shows an alignment of the predicted amino acid sequence of thelight chain variable region for antibody 8.10.3-CG4 (residues 21-129 ofSEQ ID NO: 60) to the germline V_(κ)A27, J_(κ)4 sequence (SEQ ID NO:114).

FIG. 4VV shows an alignment of the predicted amino acid sequence of theheavy chain variable region for antibody 8.10.3-CG4 (residues 20-141 ofSEQ ID NO: 94) to the germline V_(H)3-48, D_(H)1-26, J_(H)4b sequence(SEQ ID NO: 113).

FIG. 4WW shows an alignment of the predicted amino acid sequence of thelight chain variable region for antibody 9.14.4G1 (residues 23-130 ofSEQ ID NO: 28) to the germline V_(κ)O12 J_(κ)3 sequence (SEQ ID NO:103).

FIG. 4XX shows an alignment of the predicted amino acid sequence of theheavy chain variable region for antibody 9.14.4G1 (residues 20-135 ofSEQ ID NO: 102) to the germline V_(H)3-11, D_(H)7-27, J_(H)4b sequence(SEQ ID NO: 116).

FIG. 4YY shows an alignment of the predicted amino acid sequence of thelight chain variable region for antibody 8.10.3FG1 (residues 21-129 ofSEQ ID NO:32) to the germline V_(κ)A27, J_(κ)4 sequence (SEQ ID NO:114).

FIG. 4ZZ shows an alignment of the predicted amino acid sequence of theheavy chain variable region for antibody 8.10.3FG1 (residues 20-141 ofSEQ ID NO: 98) to the germline V_(H)3-48, D_(H)1-26, J_(H)4b sequence(SEQ ID NO: 113).

DETAILED DESCRIPTION OF THE INVENTION Definitions and General Techniques

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures used in connection with, and techniques of, cell andtissue culture, molecular biology, immunology, microbiology, geneticsand protein and nucleic acid chemistry and hybridization describedherein are those well known and commonly used in the art.

The methods and techniques of the present invention are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989) and Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Associates (1992), and Harlow and Lane Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1990), which are incorporated herein by reference.Enzymatic reactions and purification techniques are performed accordingto manufacturer's specifications, as commonly accomplished in the art oras described herein. The nomenclatures used in connection with, and thelaboratory procedures and techniques of, analytical chemistry, syntheticorganic chemistry, and medicinal and pharmaceutical chemistry describedherein are those well known and commonly used in the art. Standardtechniques are used for chemical syntheses, chemical analyses,pharmaceutical preparation, formulation, and delivery, and treatment ofpatients.

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

The term “polypeptide” encompasses native or artificial proteins,protein fragments and polypeptide analogs of a protein sequence. Apolypeptide may be monomeric or polymeric.

The term “isolated protein”, “isolated polypeptide” or “isolatedantibody” is a protein, polypeptide or antibody that by virtue of itsorigin or source of derivation has one to four of the following: (1) isnot associated with naturally associated components that accompany it inits native state, (2) is free of other proteins from the same species,(3) is expressed by a cell from a different species, or (4) does notoccur in nature. Thus, a polypeptide that is chemically synthesized orsynthesized in a cellular system different from the cell from which itnaturally originates will be “isolated” from its naturally associatedcomponents. A protein may also be rendered substantially free ofnaturally associated components by isolation, using protein purificationtechniques well known in the art.

Examples of isolated antibodies include an anti-M-CSF antibody that hasbeen affinity purified using M-CSF, an anti-M-CSF antibody that has beensynthesized by a hybridoma or other cell line in vitro, and a humananti-M-CSF antibody derived from a transgenic mouse.

A protein or polypeptide is “substantially pure,” “substantiallyhomogeneous,” or “substantially purified” when at least about 60 to 75%of a sample exhibits a single species of polypeptide. The polypeptide orprotein may be monomeric or multimeric. A substantially pure polypeptideor protein will typically comprise about 50%, 60%, 70%, 80% or 90% W/Wof a protein sample, more usually about 95%, and preferably will be over99% pure. Protein purity or homogeneity may be indicated by a number ofmeans well known in the art, such as polyacrylamide gel electrophoresisof a protein sample, followed by visualizing a single polypeptide bandupon staining the gel with a stain well known in the art. For certainpurposes, higher resolution may be provided by using HPLC or other meanswell known in the art for purification.

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino-terminal and/or carboxy-terminal deletion, but wherethe remaining amino acid sequence is identical to the correspondingpositions in the naturally-occurring sequence. In some embodiments,fragments are at least 5, 6, 8 or 10 amino acids long. In otherembodiments, the fragments are at least 14, at least 20, at least 50, orat least 70, 80, 90, 100, 150 or 200 amino acids long.

The term “polypeptide analog” as used herein refers to a polypeptidethat comprises a segment that has substantial identity to a portion ofan amino acid sequence and that has at least one of the followingproperties: (1) specific binding to M-CSF under suitable bindingconditions, (2) ability to inhibit M-CSF.

Typically, polypeptide analogs comprise a conservative amino acidsubstitution (or insertion or deletion) with respect to thenormally-occurring sequence. Analogs typically are at least 20 or 25amino acids long, preferably at least 50, 60, 70, 80, 90, 100, 150 or200 amino acids long or longer, and can often be as long as afull-length polypeptide.

In certain embodiments, amino acid substitutions of the antibody orantigen-binding portion thereof are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, or (4) conferor modify other physicochemical or functional properties of suchanalogs. Analogs can include various muteins of a sequence other thanthe normally-occurring peptide sequence. For example, single or multipleamino acid substitutions (preferably conservative amino acidsubstitutions) may be made in the normally-occurring sequence,preferably in the portion of the polypeptide outside the domain(s)forming intermolecular contacts.

A conservative amino acid substitution should not substantially changethe structural characteristics of the parent sequence; e.g., areplacement amino acid should not alter the anti-parallel β-sheet thatmakes up the immunoglobulin binding domain that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence. In general, glycine and prolineanalogs would not be used in an anti-parallel β-sheet. Examples ofart-recognized polypeptide secondary and tertiary structures aredescribed in Proteins, Structures and Molecular Principles (Creighton,Ed., W. H. Freeman and Company, New York (1984)); Introduction toProtein Structure (C. Branden and J. Tooze, eds., Garland Publishing,New York, N.Y. (1991)); and Thornton et al., Nature 354:105 (1991),which are each incorporated herein by reference.

Non-peptide analogs are commonly used in the pharmaceutical industry asdrugs with properties analogous to those of the template peptide. Thesetypes of non-peptide compound are termed “peptide mimetics” or“peptidomimetics.” Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber andFreidinger, TINS p. 392 (1985); and Evans et al., J. Med. Chem. 30:1229(1987), which are incorporated herein by reference. Such compounds areoften developed with the aid of computerized molecular modeling. Peptidemimetics that are structurally similar to therapeutically usefulpeptides may be used to produce an equivalent therapeutic orprophylactic effect. Generally, peptidomimetics are structurally similarto a paradigm polypeptide (i.e., a polypeptide that has a desiredbiochemical property or pharmacological activity), such as a humanantibody, but have one or more peptide linkages optionally replaced by alinkage selected from the group consisting of: —CH₂NH—, —CH₂S—,—CH₂—CH₂—, —CH═CH— (cis and trans), —COCH₂—, —CH(OH)CH₂—, and —CH₂SO—,by methods well known in the art. Systematic substitution of one or moreamino acids of a consensus sequence with a D-amino acid of the same type(e.g., D-lysine in place of L-lysine) may also be used to generate morestable peptides. In addition, constrained peptides comprising aconsensus sequence or a substantially identical consensus sequencevariation may be generated by methods known in the art (Rizo andGierasch, Ann. Rev. Biochem. 61:387 (1992), incorporated herein byreference); for example, by adding internal cysteine residues capable offorming intramolecular disulfide bridges which cyclize the peptide.

An “antibody” refers to an intact antibody or an antigen-binding portionthat competes with the intact antibody for specific binding. Seegenerally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. RavenPress, N.Y. (1989)) (incorporated by reference in its entirety for allpurposes). Antigen-binding portions may be produced by recombinant DNAtechniques or by enzymatic or chemical cleavage of intact antibodies. Insome embodiments, antigen-binding portions include Fab, Fab′, F(ab′)₂,Fd, Fv, dAb, and complementarity determining region (CDR) fragments,single-chain antibodies (scFv), chimeric antibodies, diabodies andpolypeptides that contain at least a portion of an antibody that issufficient to confer specific antigen binding to the polypeptide.

From N-terminus to C-terminus, both the mature light and heavy chainvariable domains comprise the regions FR1, CDR1, FR2, CDR2, FR3, CDR3and FR4. The assignment of amino acids to each domain is in accordancewith the definitions of Kabat, Sequences of Proteins of ImmunologicalInterest (National Institutes of Health, Bethesda, Md. (1987 and 1991)),Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987), or Chothia et al.,Nature 342:878-883 (1989).

As used herein, an antibody that is referred to by number is the same asa monoclonal antibody that is obtained from the hybridoma of the samenumber. For example, monoclonal antibody 3.8.3 is the same antibody asone obtained from hybridoma 3.8.3.

As used herein, a Fd fragment means an antibody fragment that consistsof the V_(H) and C_(H) 1 domains; an Fv fragment consists of the V_(L)and V_(H) domains of a single arm of an antibody; and a dAb fragment(Ward et al., Nature 341:544-546 (1989)) consists of a V_(H) domain.

In some embodiments, the antibody is a single-chain antibody (scFv) inwhich a V_(L) and V_(H) domains are paired to form a monovalentmolecules via a synthetic linker that enables them to be made as asingle protein chain. (Bird et al., Science 242:423-426 (1988) andHuston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988).) In someembodiments, the antibodies are diabodies, i.e., are bivalent antibodiesin which V_(H) and V_(L) domains are expressed on a single polypeptidechain, but using a linker that is too short to allow for pairing betweenthe two domains on the same chain, thereby forcing the domains to pairwith complementary domains of another chain and creating two antigenbinding sites. (See e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA90:6444-6448 (1993), and Poljak R. J. et al., Structure 2:1121-1123(1994).) In some embodiments, one or more CDRs from an antibody of theinvention may be incorporated into a molecule either covalently ornoncovalently to make it an immunoadhesin that specifically binds toM-CSF. In such embodiments, the CDR(s) may be incorporated as part of alarger polypeptide chain, may be covalently linked to anotherpolypeptide chain, or may be incorporated noncovalently.

In embodiments having one or more binding sites, the binding sites maybe identical to one another or may be different.

As used herein, the term “human antibody” means any antibody in whichthe variable and constant domain sequences are human sequences. The termencompasses antibodies with sequences derived from human genes, butwhich have been changed, e.g. to decrease possible immunogenicity,increase affinity, eliminate cysteines that might cause undesirablefolding, etc. The term emcompasses such antibodies producedrecombinantly in non-human cells, which might impart glycosylation nottypical of human cells. These antibodies may be prepared in a variety ofways, as described below.

The term “chimeric antibody” as used herein means an antibody thatcomprises regions from two or more different antibodies. In oneembodiment, one or more of the CDRs are derived from a human anti-M-CSFantibody. In another embodiment, all of the CDRs are derived from ahuman anti-M-CSF antibody. In another embodiment, the CDRs from morethan one human anti-M-CSF antibodies are combined in a chimericantibody. For instance, a chimeric antibody may comprise a CDR1 from thelight chain of a first human anti-M-CSF antibody, a CDR2 from the lightchain of a second human anti-M-CSF antibody and a CDR3 from the lightchain of a third human anti-M-CSF antibody, and the CDRs from the heavychain may be derived from one or more other anti-M-CSF antibodies.Further, the framework regions may be derived from one of the anti-M-CSFantibodies from which one or more of the CDRs are taken or from one ormore different human antibodies.

Fragments or analogs of antibodies or immunoglobulin molecules can bereadily prepared by those of ordinary skill in the art following theteachings of this specification. Preferred amino- and carboxy-termini offragments or analogs occur near boundaries of functional domains.Structural and functional domains can be identified by comparison of thenucleotide and/or amino acid sequence data to public or proprietarysequence databases. Preferably, computerized comparison methods are usedto identify sequence motifs or predicted protein conformation domainsthat occur in other proteins of known structure and/or function. Methodsto identify protein sequences that fold into a known three-dimensionalstructure are known. See Bowie et al., Science 253:164 (1991).

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIACORE® proteininteraction analysis equipment (Pharmacia Biosensor AB, Uppsala, Swedenand Piscataway, N.J.). For further descriptions, see Jonsson U. et al.,Ann. Biol. Clin. 51:19-26 (1993); Jonsson U. et al., Biotechniques11:620-627 (1991); Jonsson B. et al., J. Mol. Recognit. 8:125-131(1995); and Johnsson B. et al., Anal. Biochem. 198:268-277 (1991).

The term “K_(D)” refers to the equilibrium dissociation constant of aparticular antibody-antigen interaction.

The term “epitope” includes any protein determinant capable of specificbinding to an immunoglobulin or T-cell receptor or otherwise interactingwith a molecule. Epitopic determinants generally consist of chemicallyactive surface groupings of molecules such as amino acids or sugar sidechains and generally have specific three dimensional structuralcharacteristics, as well as specific charge characteristics. An epitopemay be “linear” or “conformational.” In a linear epitope, all of thepoints of interaction between the protein and the interacting molecule(such as an antibody) occur linearally along the primary amino acidsequence of the protein. In a conformational epitope, the points ofinteraction occur across amino acid residues on the protein that areseparated from one another. An antibody is said to specifically bind anantigen when the dissociation constant is ≤1 mM, preferably ≤100 nM andmost preferably ≤10 nM. In certain embodiments, the K_(D) is 1 pM to 500pM. In other embodiments, the K_(D) is between 500 pM to 1 μM. In otherembodiments, the K_(D) is between 1 μM to 100 nM. In other embodiments,the K_(D) is between 100 mM to 10 nM. Once a desired epitope on anantigen is determined, it is possible to generate antibodies to thatepitope, e.g., using the techniques described in the present invention.Alternatively, during the discovery process, the generation andcharacterization of antibodies may elucidate information about desirableepitopes. From this information, it is then possible to competitivelyscreen antibodies for binding to the same epitope. An approach toachieve this is to conduct cross-competition studies to find antibodiesthat competitively bind with one another, e.g., the antibodies competefor binding to the antigen. A high throughout process for “binning”antibodies based upon their cross-competition is described inInternational Patent Application No. WO 03/48731.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis(2^(nd) Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991)), which is incorporated herein by reference.

The term “polynucleotide” as referred to herein means a polymeric formof nucleotides of at least 10 bases in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide. Theterm includes single and double stranded forms.

The term “isolated polynucleotide” as used herein means a polynucleotideof genomic, cDNA, or synthetic origin or some combination thereof, whichby virtue of its origin or source of derivation, the “isolatedpolynucleotide” has one to three of the following: (1) is not associatedwith all or a portion of a polynucleotides with which the “isolatedpolynucleotide” is found in nature, (2) is operably linked to apolynucleotide to which it is not linked in nature, or (3) does notoccur in nature as part of a larger sequence.

The term “oligonucleotide” as used herein includes naturally occurring,and modified nucleotides linked together by naturally occurring andnon-naturally occurring oligonucleotide linkages. Oligonucleotides are apolynucleotide subset generally comprising a length of 200 bases orfewer. Preferably oligonucleotides are 10 to 60 bases in length and mostpreferably 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length.Oligonucleotides are usually single stranded, e.g. for primers andprobes; although oligonucleotides may be double stranded, e.g. for usein the construction of a gene mutant. Oligonucleotides of the inventioncan be either sense or antisense oligonucleotides.

The term “naturally occurring nucleotides” as used herein includesdeoxyribonucleotides and ribonucleotides. The term “modifiednucleotides” as used herein includes nucleotides with modified orsubstituted sugar groups and the like. The term “oligonucleotidelinkages” referred to herein includes oligonucleotides linkages such asphosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoroamidate, and the like. See e.g., LaPlanche et al., Nucl. AcidsRes. 14:9081 (1986); Stec et al., J. Am. Chem. Soc. 106:6077 (1984);Stein et al., Nucl. Acids Res. 16:3209 (1988); Zon et al., Anti-CancerDrug Design 6:539 (1991); Zon et al., Oligonucleotides and Analogues: APractical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford UniversityPress, Oxford England (1991)); U.S. Pat. No. 5,151,510; Uhlmann andPeyman, Chemical Reviews 90:543 (1990), the disclosures of which arehereby incorporated by reference. An oligonucleotide can include a labelfor detection, if desired.

“Operably linked” sequences include both expression control sequencesthat are contiguous with the gene of interest and expression controlsequences that act in trans or at a distance to control the gene ofinterest. The term “expression control sequence” as used herein meanspolynucleotide sequences that are necessary to effect the expression andprocessing of coding sequences to which they are ligated. Expressioncontrol sequences include appropriate transcription initiation,termination, promoter and enhancer sequences; efficient RNA processingsignals such as splicing and polyadenylation signals; sequences thatstabilize cytoplasmic mRNA; sequences that enhance translationefficiency (i.e., Kozak consensus sequence); sequences that enhanceprotein stability; and when desired, sequences that enhance proteinsecretion. The nature of such control sequences differs depending uponthe host organism; in prokaryotes, such control sequences generallyinclude promoter, ribosomal binding site, and transcription terminationsequence; in eukaryotes, generally, such control sequences includepromoters and transcription termination sequence. The term “controlsequences” is intended to include, at a minimum, all components whosepresence is essential for expression and processing, and can alsoinclude additional components whose presence is advantageous, forexample, leader sequences and fusion partner sequences.

The term “vector”, as used herein, means a nucleic acid molecule capableof transporting another nucleic acid to which it has been linked. Insome embodiments, the vector is a plasmid, i.e., a circular doublestranded DNA loop into which additional DNA segments may be ligated. Insome embodiments, the vector is a viral vector, wherein additional DNAsegments may be ligated into the viral genome. In some embodiments, thevectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., bacterial vectors having a bacterial originof replication and episomal mammalian vectors). In other embodiments,the vectors (e.g., non-episomal mammalian vectors) can be integratedinto the genome of a host cell upon introduction into the host cell, andthereby are replicated along with the host genome. Moreover, certainvectors are capable of directing the expression of genes to which theyare operatively linked. Such vectors are referred to herein as“recombinant expression vectors” (or simply, “expression vectors”).

The term “recombinant host cell” (or simply “host cell”), as usedherein, means a cell into which a recombinant expression vector has beenintroduced. It should be understood that “recombinant host cell” and“host cell” mean not only the particular subject cell but also theprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein.

The term “selectively hybridize” referred to herein means to detectablyand specifically bind. Polynucleotides, oligonucleotides and fragmentsthereof in accordance with the invention selectively hybridize tonucleic acid strands under hybridization and wash conditions thatminimize appreciable amounts of detectable binding to nonspecificnucleic acids. “High stringency” or “highly stringent” conditions can beused to achieve selective hybridization conditions as known in the artand discussed herein. One example of “high stringency” or “highlystringent” conditions is the incubation of a polynucleotide with anotherpolynucleotide, wherein one polynucleotide may be affixed to a solidsurface such as a membrane, in a hybridization buffer of 6×SSPE or SSC,50% formamide, 5×Denhardt's reagent, 0.5% SDS, 100 μg/ml denatured,fragmented salmon sperm DNA at a hybridization temperature of 42° C. for12-16 hours, followed by twice washing at 55° C. using a wash buffer of1×SSC, 0.5% SDS. See also Sambrook et al., supra, pp. 9.50-9.55.

The term “percent sequence identity” in the context of nucleic acidsequences means the percent of residues when a first contiguous sequenceis compared and aligned for maximum correspondence to a secondcontiguous sequence. The length of sequence identity comparison may beover a stretch of at least about nine nucleotides, usually at leastabout 18 nucleotides, more usually at least about 24 nucleotides,typically at least about 28 nucleotides, more typically at least about32 nucleotides, and preferably at least about 36, 48 or morenucleotides. There are a number of different algorithms known in the artwhich can be used to measure nucleotide sequence identity. For instance,polynucleotide sequences can be compared using FASTA, Gap or Bestfit,which are programs in Wisconsin Package Version 10.0, Genetics ComputerGroup (GCG), Madison, Wis. FASTA, which includes, e.g., the programsFASTA2 and FASTA3, provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol.132:185-219 (2000); Pearson, Methods Enzymol. 266:227-258 (1996);Pearson, J. Mol. Biol. 276:71-84 (1998); herein incorporated byreference). Unless otherwise specified, default parameters for aparticular program or algorithm are used. For instance, percent sequenceidentity between nucleic acid sequences can be determined using FASTAwith its default parameters (a word size of 6 and the NOPAM factor forthe scoring matrix) or using Gap with its default parameters as providedin GCG Version 6.1, herein incorporated by reference.

A reference to a nucleotide sequence encompasses its complement unlessotherwise specified. Thus, a reference to a nucleic acid having aparticular sequence should be understood to encompass its complementarystrand, with its complementary sequence.

The term “percent sequence identity” means a ratio, expressed as apercent of the number of identical residues over the number of residuescompared.

The term “substantial similarity” or “substantial sequence similarity,”when referring to a nucleic acid or fragment thereof, means that whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 85%, preferably at leastabout 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99%of the nucleotide bases, as measured by any well-known algorithm ofsequence identity, such as FASTA, BLAST or Gap, as discussed above.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, as supplied with the programs,share at least 70%, 75% or 80% sequence identity, preferably at least90% or 95% sequence identity, and more preferably at least 97%, 98% or99% sequence identity. In certain embodiments, residue positions thatare not identical differ by conservative amino acid substitutions. A“conservative amino acid substitution” is one in which an amino acidresidue is substituted by another amino acid residue having a side chainR group with similar chemical properties (e.g., charge orhydrophobicity). In general, a conservative amino acid substitution willnot substantially change the functional properties of a protein. Incases where two or more amino acid sequences differ from each other byconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well-known to thoseof skill in the art. See, e.g., Pearson, Methods Mol. Biol. 243:307-31(1994). Examples of groups of amino acids that have side chains withsimilar chemical properties include 1) aliphatic side chains: glycine,alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl sidechains: serine and threonine; 3) amide-containing side chains:asparagine and glutamine; 4) aromatic side chains: phenylalanine,tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, andhistidine; 6) acidic side chains: aspartic acid and glutamic acid; and7) sulfur-containing side chains: cysteine and methionine. Conservativeamino acids substitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine.

Alternatively, a conservative replacement is any change having apositive value in the PAM250 log-likelihood matrix disclosed in Gonnetet al., Science 256:1443-45 (1992), herein incorporated by reference. A“moderately conservative” replacement is any change having a nonnegativevalue in the PAM250 log-likelihood matrix.

Sequence identity for polypeptides, is typically measured using sequenceanalysis software. Protein analysis software matches sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG contains programs such as “Gap” and “Bestfit” whichcan be used with default parameters, as specified with the programs, todetermine sequence homology or sequence identity between closely relatedpolypeptides, such as homologous polypeptides from different species oforganisms or between a wild type protein and a mutein thereof. See,e.g., GCG Version 6.1. Polypeptide sequences also can be compared usingFASTA using default or recommended parameters, see GCG Version 6.1.(University of Wisconsin WI) FASTA (e.g., FASTA2 and FASTA3) providesalignments and percent sequence identity of the regions of the bestoverlap between the query and search sequences (Pearson, MethodsEnzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132:185-219(2000)). Another preferred algorithm when comparing a sequence of theinvention to a database containing a large number of sequences fromdifferent organisms is the computer program BLAST, especially blastp ortblastn, using default parameters, as supplied with the programs. See,e.g., Altschul et al., J. Mol. Biol. 215:403-410 (1990); Altschul etal., Nucleic Acids Res. 25:3389-402 (1997).

The length of polypeptide sequences compared for homology will generallybe at least about 16 amino acid residues, usually at least about 20residues, more usually at least about 24 residues, typically at leastabout 28 residues, and preferably more than about 35 residues. Whensearching a database containing sequences from a large number ofdifferent organisms, it is preferable to compare amino acid sequences.

As used herein, the terms “label” or “labeled” refers to incorporationof another molecule in the antibody. In one embodiment, the label is adetectable marker, e.g., incorporation of a radiolabeled amino acid orattachment to a polypeptide of biotinyl moieties that can be detected bymarked avidin (e.g., streptavidin containing a fluorescent marker orenzymatic activity that can be detected by optical or colorimetricmethods). In another embodiment, the label or marker can be therapeutic,e.g., a drug conjugate or toxin. Various methods of labelingpolypeptides and glycoproteins are known in the art and may be used.Examples of labels for polypeptides include, but are not limited to, thefollowing: radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y,⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase), chemiluminescentmarkers, biotinyl groups, predetermined polypeptide epitopes recognizedby a secondary reporter (e.g., leucine zipper pair sequences, bindingsites for secondary antibodies, metal binding domains, epitope tags),magnetic agents, such as gadolinium chelates, toxins such as pertussistoxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. In some embodiments, labels are attached by spacerarms of various lengths to reduce potential steric hindrance.

Throughout this specification and claims, the word “comprise,” orvariations such as “comprises” or “comprising,” will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

Human Anti-M-CSF Antibodies and Characterization Thereof

In one embodiment, the invention provides humanized anti-M-CSFantibodies. In another embodiment, the invention provides humananti-M-CSF antibodies. In some embodiments, human anti-M-CSF antibodiesare produced by immunizing a non-human transgenic animal, e.g., arodent, whose genome comprises human immunoglobulin genes so that therodent produces human antibodies.

An anti-M-CSF antibody of the invention can comprise a human kappa or ahuman lamda light chain or an amino acid sequence derived therefrom. Insome embodiments comprising a kappa light chain, the light chainvariable domain (V_(L)) is encoded in part by a human V_(κ)O12, V_(κ)L2,V_(κ)L5, V_(κ)A27 or V_(κ)B3 gene and a J_(κ)1, J_(κ)2, J_(κ)3, orJ_(κ)4 gene. In particular embodiments of the invention, the light chainvariable domain is encoded by V_(κ)O12/Jκ3, V_(κ)L2/Jκ3, V_(κ)L5/Jκ3,V_(κ)L5/Jκ4, V_(κ)A27/Jκ4 or V_(κ)B3/Jκ1 gene.

In some embodiments, the V_(L) of the M-CSF antibody comprises one ormore amino acid substitutions relative to the germline amino acidsequence. In some embodiments, the V_(L) of the anti-M-CSF antibodycomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutionsrelative to the germline amino acid sequence. In some embodiments, oneor more of those substitutions from germline is in the CDR regions ofthe light chain. In some embodiments, the amino acid substitutionsrelative to germline are at one or more of the same positions as thesubstitutions relative to germline in any one or more of the V_(L) ofantibodies 252, 88, 100, 3.8.3, 2.7.3, 1.120.1, 9.14.4I, 8.10.3F,9.7.2IF, 9.14.4, 8.10.3, 9.7.2, 9.7.2C-Ser, 9.14.4C-Ser, 8.10.3C-Ser,8.10.3-CG2, 9.7.2-CG2, 9.7.2-CG4, 9.14.4-CG2, 9.14.4-CG4, 9.14.4-Ser,9.7.2-Ser, 8.10.3-Ser, 8.10.3-CG4, 8.10.3FG1 or 9.14.4G1. For example,the V_(L) of the anti-M-CSF antibody may contain one or more amino acidsubstitutions compared to germline found in the V_(L) of antibody 88,and other amino acid substitutions compared to germline found in theV_(L) of antibody 252 which utilizes the same V_(K) gene as antibody 88.In some embodiments, the amino acid changes are at one or more of thesame positions but involve a different mutation than in the referenceantibody.

In some embodiments, amino acid changes relative to germline occur atone or more of the same positions as in any of the V_(L) of antibodies252, 88, 100, 3.8.3, 2.7.3, 1.120.1, 9.14.4I, 8.10.3F, 9.7.2IF, 9.14.4,8.10.3, 9.7.2, 9.7.2C-Ser, 9.14.4C-Ser, 8.10.3C-Ser, 8.10.3-CG2,9.7.2-CG2, 9.7.2-CG4, 9.14.4-CG2, 9.14.4-CG4, 9.14.4-Ser, 9.7.2-Ser,8.10.3-Ser, 8.10.3-CG4, 8.10.3FG1 or 9.14.4G1, but the changes mayrepresent conservative amino acid substitutions at such position(s)relative to the amino acid in the reference antibody. For example, if aparticular position in one of these antibodies is changed relative togermline and is glutamate, one may substitute aspartate at thatposition. Similarly, if an amino acid substitution compared to germlineis serine, one may substitute threonine for serine at that position.Conservative amino acid substitutions are discussed supra.

In some embodiments, the light chain of the human anti-M-CSF antibodycomprises the amino acid sequence that is the same as the amino acidsequence of the V_(L) of antibody 252 (SEQ ID NO: 4), 88 (SEQ ID NO: 8),100 (SEQ ID NO: 12), 3.8.3 (SEQ ID NO: 16), 2.7.3 (SEQ ID NO: 20),1.120.1 (SEQ ID NO: 24), 9.14.4I (SEQ ID NO: 28), 8.10.3F (SEQ ID NO:32), 9.7.2IF (SEQ ID NO: 36), 9.14.4 (SEQ ID NO: 28), 8.10.3 (SEQ ID NO:44), 9.7.2 (SEQ ID NO: 48), 9.7.2C-Ser (SEQ ID NO: 52), 9.14.4C-Ser (SEQID NO: 56), 8.10.3C-Ser (SEQ ID NO: 60), 8.10.3-CG2 (SEQ ID NO: 60),9.7.2-CG2 (SEQ ID NO: 52), 9.7.2-CG4 (SEQ ID NO: 52), 9.14.4-CG2 (SEQ IDNO: 56), 9.14.4-CG4 (SEQ ID NO: 56), 9.14.4-Ser (SEQ ID NO: 28),9.7.2-Ser (SEQ ID NO: 48), 8.10.3-Ser (SEQ ID NO: 44), 8.10.3-CG4 (SEQID NO: 60) 8.10.3FG1 (SEQ ID NO: 32) or 9.14.4G1 (SEQ ID NO: 28), orsaid amino acid sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10conservative amino acid substitutions and/or a total of up to 3non-conservative amino acid substitutions. In some embodiments, thelight chain comprises the amino acid sequence from the beginning of theCDR1 to the end of the CDR3 of any one of the foregoing antibodies.

In some embodiments, the light chain of the anti-M-CSF antibodycomprises at least the light chain CDR1, CDR2 or CDR3 of a germline orantibody sequence, as described herein. In another embodiment, the lightchain may comprise a CDR1, CDR2 or CDR3 regions of an antibodyindependently selected from 252, 88, 100, 3.8.3, 2.7.3, 1.120.1,9.14.4I, 8.10.3F, 9.7.2IF, 9.14.4, 8.10.3, 9.7.2, 9.7.2C-Ser,9.14.4C-Ser, 8.10.3C-Ser, 8.10.3-CG2, 9.7.2-CG2, 9.7.2-CG4, 9.14.4-CG2,9.14.4-CG4, 9.14.4-Ser, 9.7.2-Ser, 8.10.3-Ser, 8.10.3-CG4, 8.10.3FG1 or9.14.4G1, or CDR regions each having less than 4 or less than 3conservative amino acid substitutions and/or a total of three or fewernon-conservative amino acid substitutions. In other embodiments, thelight chain of the anti-M-CSF antibody comprises the light chain CDR1,CDR2 or CDR3, each of which are independently selected from the CDR1,CDR2 and CDR3 regions of an antibody having a light chain variableregion comprising the amino acid sequence of the V_(L) region selectedfrom SEQ ID NOS: 4, 8, 12, 16, 20, 24, 28, 32, 36, 44, 48, 52, 56 or 60,or encoded by a nucleic acid molecule encoding the V_(L) region selectedfrom SEQ ID NOS: 3, 7, 11, 27, 31, 35, 43 or 47. The light chain of theanti-M-CSF antibody may comprise the CDR1, CDR2 and CDR3 regions of anantibody comprising the amino acid sequence of the V_(L) region selectedfrom 252, 88, 100, 3.8.3, 2.7.3, 1.120.1, 9.14.4I, 8.10.3F, 9.7.2IF,9.14.4, 8.10.3, 9.7.2, 9.7.2C-Ser, 9.14.4C-Ser, 8.10.3C-Ser, 8.10.3-CG2,9.7.2-CG2, 9.7.2-CG4, 9.14.4-CG2, 9.14.4-CG4, 9.14.4-Ser, 9.7.2-Ser,8.10.3-Ser, 8.10.3-CG4, 8.10.3FG1 or 9.14.4G1 or SEQ ID NOS: 4, 8, 12,16, 20, 24, 28, 32, 36, 44, 48, 52, 56 or 60.

In some embodiments, the light chain comprises the CDR1, CDR2 and CDR3regions of antibody 252, 88, 100, 3.8.3, 2.7.3, 1.120.1, 9.14.4I,8.10.3F, 9.7.2IF, 9.14.4, 8.10.3, 9.7.2, 9.7.2C-Ser, 9.14.4C-Ser,8.10.3C-Ser, 8.10.3-CG2, 9.7.2-CG2, 9.7.2-CG4, 9.14.4-CG2, 9.14.4-CG4,9.14.4-Ser, 9.7.2-Ser, 8.10.3-Ser, 8.10.3-CG4, 8.10.3FG1 or 9.14.4G1, orsaid CDR regions each having less than 4 or less than 3 conservativeamino acid substitutions and/or a total of three or fewernon-conservative amino acid substitutions.

With regard to the heavy chain, in some embodiments, the variable regionof the heavy chain amino acid sequence is encoded in part by a humanV_(H)3-11, V_(H)3-23, V_(H)3-7, V_(H)1-18, V_(H)3-33, V_(H)3-48 gene anda J_(H)4, J_(H)6, J_(H)4b, or J_(H)6b gene. In a particular embodimentof the invention, the heavy chain variable region is encoded byV_(H)3-11/D_(H)7-27/J_(H)6, V_(H)3-7/D_(H)6-13/J_(H)4,V_(H)3-23/D_(H)1-26/J_(H)4, V_(H)3-11/D_(H)7-27/J_(H)4,V_(H)3-33/D_(H)1-26/J_(H)4, V_(H)1-18/D_(H)4-23/J_(H)4,V_(H)3-11/D_(H)7-27/J_(H)4b, V_(H)3-48/D_(H)1-26/J_(H)4b,V_(H)3-11/D_(H)6-13/J_(H)6b, V_(H)3-11/D_(H)7-27/J_(H)4b,V_(H)3-48/D_(H)1-6/J_(H)4b, or V_(H)3-11/D_(H)6-13/J_(H)6b gene. In someembodiments, the V_(H) of the anti-M-CSF antibody contains one or moreamino acid substitutions, deletions or insertions (additions) relativeto the germline amino acid sequence. In some embodiments, the variabledomain of the heavy chain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, or 18 mutations from the germline amino acidsequence. In some embodiments, the mutation(s) are non-conservativesubstitutions compared to the germline amino acid sequence. In someembodiments, the mutations are in the CDR regions of the heavy chain. Insome embodiments, the amino acid changes are made at one or more of thesame positions as the mutations from germline in any one or more of theV_(H) of antibodies 252, 88, 100, 3.8.3, 2.7.3, 1.120.1, 9.14.4I,8.10.3F, 9.7.2IF, 9.14.4, 8.10.3, 9.7.2, 9.7.2C-Ser, 9.14.4C-Ser,8.10.3C-Ser, 8.10.3-CG2, 9.7.2-CG2, 9.7.2-CG4, 9.14.4-CG2, 9.14.4-CG4,9.14.4-Ser, 9.7.2-Ser, 8.10.3-Ser, 8.10.3-CG4, 8.10.3FG1 or 9.14.4G1. Inother embodiments, the amino acid changes are at one or more of the samepositions but involve a different mutation than in the referenceantibody.

In some embodiments, the heavy chain comprises an amino acid sequence ofthe variable domain (V_(H)) of antibody 252 (SEQ ID NO: 2), 88 (SEQ IDNO: 6), 100 (SEQ ID NO: 10), 3.8.3 (SEQ ID NO: 14), 2.7.3 (SEQ. ID NO:18), 1.120.1 (SEQ. ID NO: 22), 9.14.4I (SEQ ID NO: 26), 8.10.3F (SEQ IDNO: 30), 9.7.2IF (SEQ ID NO: 34), 9.14.4 (SEQ ID NO: 38), 8.10.3 (SEQ IDNO: 30), 9.7.2 (SEQ ID NO: 46), 9.7.2C-Ser (SEQ ID NO: 50), 9.14.4C-Ser(SEQ ID NO: 54), 8.10.3C-Ser (SEQ ID NO: 58), 8.10.3-CG2 (SEQ ID NO:62), 9.7.2-CG2 (SEQ ID NO: 66), 9.7.2-CG4 (SEQ ID NO: 70), 9.14.4-CG2(SEQ ID NO: 74), 9.14.4-CG4 (SEQ ID NO: 78), 9.14.4-Ser (SEQ ID NO: 82),9.7.2-Ser (SEQ ID NO: 86), 8.10.3-Ser (SEQ ID NO: 90) 8.10.3-CG4 (SEQ IDNO: 94), 8.10.3FG1 (SEQ ID NO: 98) or 9.14.4G1 (SEQ ID NO: 102), or saidamino acid sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10conservative amino acid substitutions and/or a total of up to 3non-conservative amino acid substitutions. In some embodiments, theheavy chain comprises the amino acid sequence from the beginning of theCDR1 to the end of the CDR3 of any one of the foregoing antibodies.

In some embodiments, the heavy chain comprises the heavy chain CDR1,CDR2 and CDR3 regions of antibody 252, 88, 100, 3.8.3, 2.7.3, 1.120.1,9.14.4I, 8.10.3F, 9.7.2IF, 9.14.4, 8.10.3, 9.7.2, 9.7.2C-Ser,9.14.4C-Ser, 8.10.3C-Ser, 8.10.3-CG2, 9.7.2-CG2, 9.7.2-CG4, 9.14.4-CG2,9.14.4-CG4, 9.14.4-Ser, 9.7.2-Ser, 8.10.3-Ser, 8.10.3-CG4, 8.10.3FG1 or9.14.4G1, or said CDR regions each having less than 8, less than 6, lessthan 4, or less than 3 conservative amino acid substitutions and/or atotal of three or fewer non-conservative amino acid substitutions.

In some embodiments, the heavy chain comprises a germline or antibodyCDR3, as described above, of an antibody sequence as described herein,and may also comprise the CDR1 and CDR2 regions of a germline sequence,or may comprise a CDR1 and CDR2 of an antibody sequence, each of whichare independently selected from an antibody comprising a heavy chain ofan antibody selected from 252, 88, 100, 3.8.3, 2.7.3, 1.120.1, 9.14.4I,8.10.3F, 9.7.2IF, 9.14.4, 8.10.3, 9.7.2, 9.7.2C-Ser, 9.14.4C-Ser,8.10.3C-Ser, 8.10.3-CG2, 9.7.2-CG2, 9.7.2-CG4, 9.14.4-CG2, 9.14.4-CG4,9.14.4-Ser, 9.7.2-Ser, 8.10.3-Ser, 8.10.3-CG4, 8.10.3FG1 or 9.14.4G1. Inanother embodiment, the heavy chain comprises a CDR3 of an antibodysequence as described herein, and may also comprise the CDR1 and CDR2regions, each of which are independently selected from a CDR1 and CDR2region of a heavy chain variable region comprising an amino acidsequence of the V_(H) region selected from SEQ ID NOS: 2, 6, 10, 14, 18,22, 26, 30, 34, 38, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94,98 or 102, or encoded by a nucleic acid sequence encoding the V_(H)region selected from SEQ ID NOS: 1, 5, 9, 25, 29, 33, 37, 45, 97 or 101.In another embodiment, the antibody comprises a light chain as disclosedabove and a heavy chain as disclosed above.

One type of amino acid substitution that may be made is to change one ormore cysteines in the antibody, which may be chemically reactive, toanother residue, such as, without limitation, alanine or serine. In oneembodiment, there is a substitution of a non-canonical cysteine. Thesubstitution can be in a framework region of a variable domain or in theconstant domain of an antibody. In another embodiment, the cysteine isin a non-canonical region of the antibody.

Another type of amino acid substitution that may be made is to removeany potential proteolytic sites in the antibody, particularly those thatare in a CDR or framework region of a variable domain or in the constantdomain of an antibody. Substitution of cysteine residues and removal ofproteolytic sites may decrease the risk of any heterogeneity in theantibody product and thus increase its homogeneity. Another type ofamino acid substitution is elimination of asparagine-glycine pairs,which form potential deamidation sites, by altering one or both of theresidues.

In some embodiments, the C-terminal lysine of the heavy chain of theanti-M-CSF antibody of the invention is not present (Lewis D. A., etal., Anal. Chem, 66(5): 585-95 (1994)). In various embodiments of theinvention, the heavy and light chains of the anti-M-CSF antibodies mayoptionally include a signal sequence.

In one aspect, the invention relates to inhibiting human anti-M-CSFmonoclonal antibodies and the cell lines engineered to produce them.Table 1 lists the sequence identifiers (SEQ ID NOS) of the nucleic acidsthat encode the variable region of the heavy and light chains and thecorresponding predicted amino acid sequences for the monoclonalantibodies: 252, 88, 100, 3.8.3, 2.7.3, 1.120.1, 9.14.4I, 8.10.3F,9.7.2IF, 9.14.4, 8.10.3 and 9.7.2. Additional variant antibodies9.7.2C-Ser, 9.14.4C-Ser, 8.10.3C-Ser, 8.10.3-CG2, 9.7.2-CG2, 9.7.2-CG4,9.14.4-CG2, 9.14.4-CG4, 9.14.4-Ser, 9.7.2-Ser, 8.10.3-Ser, 8.10.3-CG48.10.3FG1 or 9.14.4G1 could be made by methods known to one skilled inthe art.

TABLE 1 HUMAN ANTI-M-CSF ANTIBODIES SEQUENCE IDENTIFIER (SEQ ID NOS:)Full Length Heavy Light MAb DNA Protein DNA Protein 252 1 2 3 4  88 5 67 8 100 9 10 11 12 3.8.3 14 16 2.7.3 18 20 1.120.1 22 24 9.14.4I 25 2627 28 9.14.4 37 38 27 28 9.14.4C-Ser 54 56 9.14.4-CG2 74 56 9.14.4-CG478 56 9.14.4-Ser 82 27 28 9.14.4-G1 101 102 27 28 8.10.3F 29 30 31 328.10.3 29 30 43 44 8.10.3C-Ser 58 60 8.10.3-CG2 62 60 8.10.3-Ser 90 4344 8.10.3-CG4 94 60 8.10.3FG1 97 98 31 32 9.7.2IF 33 34 35 36 9.7.2 4546 47 48 9.7.2C-Ser 50 52 9.7.2-CG2 66 52 9.7.2-CG4 70 52 9.7.2-Ser 8647 48Class and Subclass of Anti-M-CSF Antibodies

The class and subclass of anti-M-CSF antibodies may be determined by anymethod known in the art. In general, the class and subclass of anantibody may be determined using antibodies that are specific for aparticular class and subclass of antibody. Such antibodies arecommercially available. The class and subclass can be determined byELISA, or Western Blot as well as other techniques. Alternatively, theclass and subclass may be determined by sequencing all or a portion ofthe constant domains of the heavy and/or light chains of the antibodies,comparing their amino acid sequences to the known amino acid sequencesof various class and subclasses of immunoglobulins, and determining theclass and subclass of the antibodies.

In some embodiments, the anti-M-CSF antibody is a monoclonal antibody.The anti-M-CSF antibody can be an IgG, an IgM, an IgE, an IgA, or an IgDmolecule. In preferred embodiments, the anti-M-CSF antibody is an IgGand is an IgG1, IgG2, IgG3 or IgG4 subclass. In other preferredembodiments, the antibody is subclass IgG2 or IgG4. In another preferredembodiment, the antibody is subclass IgG1.

Species and Molecular Selectivity

In another aspect of the invention, the anti-M-CSF antibodiesdemonstrate both species and molecule selectivity. In some embodiments,the anti-M-CSF antibody binds to human, cynomologus monkey and mouseM-CSF. Following the teachings of the specification, one may determinethe species selectivity for the anti-M-CSF antibody using methods wellknown in the art. For instance, one may determine the speciesselectivity using Western blot, FACS, ELISA, RIA, a cell proliferationassay, or a M-CSF receptor binding assay. In a preferred embodiment, onemay determine the species selectivity using a cell proliferation assayor ELISA. In some embodiments, the anti-M-CSF antibodies bind to humansecreted isoforms of M-CSF and membrane bound isoforms of M-CSF.

In another embodiment, the anti-M-CSF antibody has a selectivity forM-CSF that is at least 100 times greater than its selectivity forGM-/G-CSF. In some embodiments, the anti-M-CSF antibody does not exhibitany appreciable specific binding to any other protein other than M-CSF.One can determine the selectivity of the anti-M-CSF antibody for M-CSFusing methods well known in the art following the teachings of thespecification. For instance one can determine the selectivity usingWestern blot, FACS, ELISA, or RIA.

Identification of M-CSF Epitopes Recognized by Anti-M-CSF Antibodies

The invention provides a human anti-M-CSF monoclonal antibody that bindsto M-CSF and competes with, cross-competes with and/or binds the sameepitope and/or binds to M-CSF with the same K_(D) as (a) an antibodyselected from 252, 88, 100, 3.8.3, 2.7.3, 1.120.1, 9.14.4I, 8.10.3F,9.7.2IF, 9.14.4, 8.10.3, 9.7.2, 9.7.2C-Ser, 9.14.4C-Ser, 8.10.3C-Ser,8.10.3-CG2, 9.7.2-CG2, 9.7.2-CG4, 9.14.4-CG2, 9.14.4-CG4, 9.14.4-Ser,9.7.2-Ser, 8.10.3-Ser, 8.10.3-CG4, 8.10.3FG1 or 9.14.4G1; (b) anantibody that comprises a heavy chain variable region having an aminoacid sequence of SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 46,50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98 or 102; (c) anantibody that comprises a light chain variable region having an aminoacid sequence of SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 32, 36, 44, 48,52, 56 or 60; (d) an antibody that comprises both a heavy chain variableregion as defined in (b) and a light chain variable region as defined in(c).

One can determine whether an antibody binds to the same epitope,competes for binding with, cross competes for binding with or has thesame K_(D) an anti-M-CSF antibody by using methods known in the art. Inone embodiment, one allows the anti-M-CSF antibody of the invention tobind to M-CSF under saturating conditions and then measures the abilityof the test antibody to bind to M-CSF. If the test antibody is able tobind to M-CSF at the same time as the anti-M-CSF antibody, then the testantibody binds to a different epitope as the anti-M-CSF antibody.However, if the test antibody is not able to bind to M-CSF at the sametime, then the test antibody binds to the same epitope, an overlappingepitope, or an epitope that is in close proximity to the epitope boundby the human anti-M-CSF antibody. This experiment can be performed usingELISA, RIA, or FACS. In a preferred embodiment, the experiment isperformed using BIACORE® protein interaction analysis equipment.

Binding Affinity of Anti-M-CSF Antibodies to M-CSF

In some embodiments of the invention, the anti-M-CSF antibodies bind toM-CSF with high affinity. In some embodiments, the anti-M-CSF antibodybinds to M-CSF with a K_(D) of 1×10⁻⁷M or less. In other preferredembodiments, the antibody binds to M-CSF with a K_(D) of 1×10⁻⁸ M,1×10⁻⁹ M, 1×10⁻¹⁰ M, 1×10⁻¹¹ M, 1×10⁻¹²M or less. In certainembodiments, the K_(D) is 1 pM to 500 pM. In other embodiments, theK_(D) is between 500 pM to 1 μM. In other embodiments, the K_(D) isbetween 1 μM to 100 nM. In other embodiments, the K_(D) is between 100mM to 10 nM. In an even more preferred embodiment, the antibody binds toM-CSF with substantially the same K_(D) as an antibody selected from252, 88, 100, 3.8.3, 2.7.3, 1.120.1, 9.14.4I, 8.10.3F, 9.7.2IF, 9.14.4,8.10.3, 9.7.2, 9.7.2C-Ser, 9.14.4C-Ser, 8.10.3C-Ser, 8.10.3-CG2,9.7.2-CG2, 9.7.2-CG4, 9.14.4-CG2, 9.14.4-CG4, 9.14.4-Ser, 9.7.2-Ser,8.10.3-Ser, 8.10.3-CG4, 8.10.3FG1 or 9.14.4G1. In another preferredembodiment, the antibody binds to M-CSF with substantially the sameK_(D) as an antibody that comprises a CDR2 of a light chain, and/or aCDR3 of a heavy chain from an antibody selected from 252, 88, 100,3.8.3, 2.7.3, 1.120.1, 9.14.4I, 8.10.3F, 9.7.2IF, 9.14.4, 8.10.3, 9.7.2,9.7.2C-Ser, 9.14.4C-Ser, 8.10.3C-Ser, 8.10.3-CG2, 9.7.2-CG2, 9.7.2-CG4,9.14.4-CG2, 9.14.4-CG4, 9.14.4-Ser, 9.7.2-Ser, 8.10.3-Ser, 8.10.3-CG4,8.10.3FG1 or 9.14.4G1. In still another preferred embodiment, theantibody binds to M-CSF with substantially the same K_(D) as an antibodythat comprises a heavy chain variable region having an amino acidsequence of SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 46, 50, 54,58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98 or 102, or that comprises alight chain variable region having an amino acid sequence of SEQ ID NO:4, 8, 12, 16, 20, 24, 28, 32, 36, 44, 48, 52, 56 or 60. In anotherpreferred embodiment, the antibody binds to M-CSF with substantially thesame K_(D) as an antibody that comprises a CDR2, and may optionallycomprise a CDR1 and/or CDR3, of a light chain variable region having anamino acid sequence of the V_(L) region of SEQ ID NO: 4, 8, 12, 16, 20,24, 28, 32, 36, 44, 48, 52, 56 or 60, or that comprises a CDR3, and mayoptionally comprise a CDR1 and/or CDR2, of a heavy chain variable regionhaving an amino acid sequence of the V_(H) region of SEQ ID NO: 2, 6,10, 14, 18, 22, 26, 30, 34, 38, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82,86, 90, 94, 98 or 102.

In some embodiments, the anti-M-CSF antibody has a low dissociationrate. In some embodiments, the anti-M-CSF antibody has an k_(off) of2.0×10⁻⁴ s⁻¹ or lower. In other preferred embodiments, the antibodybinds to M-CSF with a k_(off) of 2.0×10⁻⁵ or a k_(off) 2.0×10⁻⁶ s⁻¹ orlower. In some embodiments, the k_(off) is substantially the same as anantibody described herein, such as an antibody selected from 252, 88,100, 3.8.3, 2.7.3, 1.120.1, 9.14.4I, 8.10.3F, 9.7.2IF, 9.14.4, 8.10.3,9.7.2, 9.7.2C-Ser, 9.14.4C-Ser, 8.10.3C-Ser, 8.10.3-CG2, 9.7.2-CG2,9.7.2-CG4, 9.14.4-CG2, 9.14.4-CG4, 9.14.4-Ser, 9.7.2-Ser, 8.10.3-Ser,8.10.3-CG4, 8.10.3FG1 or 9.14.4G1. In some embodiments, the antibodybinds to M-CSF with substantially the same k_(off) as an antibody thatcomprises (a) a CDR3, and may optionally comprise a CDR1 and/or CDR2, ofa heavy chain of an antibody selected from 252, 88, 100, 3.8.3, 2.7.3,1.120.1, 9.14.4I, 8.10.3F, 9.7.2IF, 9.14.4, 8.10.3, 9.7.2, 9.7.2C-Ser,9.14.4C-Ser, 8.10.3C-Ser, 8.10.3-CG2, 9.7.2-CG2, 9.7.2-CG4, 9.14.4-CG2,9.14.4-CG4, 9.14.4-Ser, 9.7.2-Ser, 8.10.3-Ser, 8.10.3-CG4, 8.10.3FG1 or9.14.4G1; or (b) a CDR2, and may optionally comprise a CDR1 and/or CDR3,of a light chain from an antibody selected from 252, 88, 100, 3.8.3,2.7.3, 1.120.1, 9.14.4I, 8.10.3F, 9.7.2IF, 9.14.4, 8.10.3, 9.7.2,9.7.2C-Ser, 9.14.4C-Ser, 8.10.3C-Ser, 8.10.3-CG2, 9.7.2-CG2, 9.7.2-CG4,9.14.4-CG2, 9.14.4-CG4, 9.14.4-Ser, 9.7.2-Ser, 8.10.3-Ser, 8.10.3-CG4,8.10.3FG1 or 9.14.4G1. In some embodiments, the antibody binds to M-CSFwith substantially the same k_(off) as an antibody that comprises aheavy chain variable region having an amino acid sequence of SEQ ID NO:2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 46, 50, 54, 58, 62, 66, 70, 74,78, 82, 86, 90, 94, 98 or 102; or that comprises a light chain variableregion having an amino acid sequence of SEQ ID NO: 4, 8, 12, 16, 20, 24,28, 32, 36, 44, 48, 52, 56 or 60; In another preferred embodiment, theantibody binds to M-CSF with substantially the same k_(off) as anantibody that comprises a CDR2, and may optionally comprise a CDR1and/or CDR3, of a light chain variable region having an amino acidsequence of SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 32, 36, 44, 48, 52, 56or 60; or a CDR3, and may optionally comprise a CDR1 and/or CDR2, of aheavy chain variable region having an amino acid sequence of SEQ ID NO:2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 46, 50, 54, 58, 62, 66, 70, 74,78, 82, 86, 90, 94, 98 or 102.

The binding affinity and dissociation rate of an anti-M-CSF antibody toa M-CSF can be determined by methods known in the art. The bindingaffinity can be measured by competitive ELISAs, RIAs, surface plasmonresonance (e.g., by using BIACORE® protein interaction analysisequipment). The dissociation rate can be measured by surface plasmonresonance. Preferably, the binding affinity and dissociation rate ismeasured by surface plasmon resonance. More preferably, the bindingaffinity and dissociation rate are measured using BIACORE® proteininteraction analysis equipment. Example VI exemplifies a method fordetermining affinity constants of anti-M-CSF monoclonal antibodies byBIACORE® protein interaction analysis equipment.

Inhibition of M-CSF Activity by Anti-M-CSF Antibody

Inhibition of M-CSF Binding to c-Fms

In another embodiment, the invention provides an anti-M-CSF antibodythat inhibits the binding of a M-CSF to c-fms receptor and blocks orprevents activation of c-fms. In an preferred embodiment, the M-CSF ishuman. In another preferred embodiment, the anti-M-CSF antibody is ahuman antibody. The IC₅₀ can be measured by ELISA, RIA, and cell basedassays such as a cell proliferation assay, a whole blood monocyte shapechange assay, or a receptor binding inhibition assay. In one embodiment,the antibody or portion thereof inhibits cell proliferation with an IC₅₀of no more than 8.0×10⁻⁷M, preferably no more than 3×10⁻⁷ M, or morepreferably no more than 8×10⁻⁸ M as measured by a cell proliferationassay. In another embodiment, the IC₅₀ as measured by a monocyte shapechange assay is no more than 2×10⁻⁶ M, preferably no more than 9.0×10⁻⁷M, or more preferably no more than 9×10⁻⁸ M. In another preferredembodiment, the IC₅₀ as measured by a receptor binding assay is no morethan 2×10⁻⁶ M, preferably no more than 8.0×10⁻⁷ M, or more preferably nomore than 7.0×10⁻⁸ M. Examples III, IV, and V exemplify various types ofassays.

In another aspect anti-M-CSF antibodies of the invention inhibitmonocyte/macrophage cell proliferation in response to a M-CSF by atleast 20%, more preferably 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 85%,90%, 95% or 100% compared to the proliferation of cell in the absence ofantibody.

Methods of Producing Antibodies and Antibody Producing Cell Lines

Immunization

In some embodiments, human antibodies are produced by immunizing anon-human animal comprising in its genome some or all of humanimmunoglobulin heavy chain and light chain loci with a M-CSF antigen. Ina preferred embodiment, the non-human animal is a XENOMOUSE® transgenicmouse that makes human antibodies (Abgenix Inc., Fremont, Calif.).Another non-human animal that may be used is a transgenic mouse producedby Medarex (Medarex, Inc., Princeton, N.J.).

XENOMOUSE® transgenic mice that make human antibodies are engineeredmouse strains that comprise large fragments of human immunoglobulinheavy chain and light chain loci and are deficient in mouse antibodyproduction. See, e.g., Green et al., Nature Genetics 7:13-21 (1994) andU.S. Pat. Nos. 5,916,771, 5,939,598, 5,985,615, 5,998,209, 6,075,181,6,091,001, 6,114,598, 6,130,364, 6,162,963 and 6,150,584. See also WO91/10741, WO 94/02602, WO 96/34096, WO 96/33735, WO 98/16654, WO98/24893, WO 98/50433, WO 99/45031, WO 99/53049, WO 00/09560, and WO00/037504.

In another aspect, the invention provides a method for making anti-M-CSFantibodies from non-human, non-mouse animals by immunizing non-humantransgenic animals that comprise human immunoglobulin loci with a M-CSFantigen. One can produce such animals using the methods described in theabove-cited documents. The methods disclosed in these documents can bemodified as described in U.S. Pat. No. 5,994,619. U.S. Pat. No.5,994,619 describes methods for producing novel cultural inner cell mass(CICM) cells and cell lines, derived from pigs and cows, and transgenicCICM cells into which heterologous DNA has been inserted. CICMtransgenic cells can be used to produce cloned transgenic embryos,fetuses, and offspring. The '619 patent also describes the methods ofproducing the transgenic animals, that are capable of transmitting theheterologous DNA to their progeny. In preferred embodiments, thenon-human animals are rats, sheep, pigs, goats, cattle or horses.

XENOMOUSE® transgenic mice that make human antibodies produce anadult-like human repertoire of fully human antibodies and generateantigen-specific human antibodies. In some embodiments, the XENOMOUSE®transgenic mice that make human antibodies contain approximately 80% ofthe human antibody V gene repertoire through introduction of megabasesized, germline configuration yeast artificial chromosome (YAC)fragments of the human heavy chain loci and kappa light chain loci. Inother embodiments, XENOMOUSE® transgenic mice that make human antibodiesfurther contain approximately all of the lambda light chain locus. SeeMendez et al., Nature Genetics 15:146-156 (1997), Green and Jakobovits,J. Exp. Med. 188:483-495 (1998), and WO 98/24893, the disclosures ofwhich are hereby incorporated by reference.

In some embodiments, the non-human animal comprising humanimmunoglobulin genes are animals that have a human immunoglobulin“minilocus”. In the minilocus approach, an exogenous Ig locus ismimicked through the inclusion of individual genes from the Ig locus.Thus, one or more V_(H) genes, one or more D_(H) genes, one or moreJ_(H) genes, a mu constant domain, and a second constant domain(preferably a gamma constant domain) are formed into a construct forinsertion into an animal. This approach is described, inter alia, inU.S. Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425,5,661,016, 5,770,429, 5,789,650, 5,814,318, 5,591,669, 5,612,205,5,721,367, 5,789,215, and 5,643,763, hereby incorporated by reference.

In another aspect, the invention provides a method for making humanizedanti-M-CSF antibodies. In some embodiments, non-human animals areimmunized with a M-CSF antigen as described below under conditions thatpermit antibody production. Antibody-producing cells are isolated fromthe animals, fused with myelomas to produce hybridomas, and nucleicacids encoding the heavy and light chains of an anti-M-CSF antibody ofinterest are isolated. These nucleic acids are subsequently engineeredusing techniques known to those of skill in the art and as describedfurther below to reduce the amount of non-human sequence, i.e., tohumanize the antibody to reduce the immune response in humans

In some embodiments, the M-CSF antigen is isolated and/or purifiedM-CSF. In a preferred embodiment, the M-CSF antigen is human M-CSF. Insome embodiments, the M-CSF antigen is a fragment of M-CSF. In someembodiments, the M-CSF fragment is the extracellular domain of M-CSF. Insome embodiments, the M-CSF fragment comprises at least one epitope ofM-CSF. In other embodiments, the M-CSF antigen is a cell that expressesor overexpresses M-CSF or an immunogenic fragment thereof on itssurface. In some embodiments, the M-CSF antigen is a M-CSF fusionprotein. M-CSF can be purified from natural sources using knowntechniques. Recombinant M-CSF is commercially available.

Immunization of animals can be by any method known in the art. See,e.g., Harlow and Lane, Antibodies: A Laboratory Manual, New York: ColdSpring Harbor Press, 1990. Methods for immunizing non-human animals suchas mice, rats, sheep, goats, pigs, cattle and horses are well known inthe art. See, e.g., Harlow and Lane, supra, and U.S. Pat. No. 5,994,619.In a preferred embodiment, the M-CSF antigen is administered with anadjuvant to stimulate the immune response. Exemplary adjuvants includecomplete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides) orISCOM (immunostimulating complexes). Such adjuvants may protect thepolypeptide from rapid dispersal by sequestering it in a local deposit,or they may contain substances that stimulate the host to secretefactors that are chemotactic for macrophages and other components of theimmune system. Preferably, if a polypeptide is being administered, theimmunization schedule will involve two or more administrations of thepolypeptide, spread out over several weeks. Example I exemplifies amethod for producing anti-M-CSF monoclonal antibodies in XENOMOUSE®transgenic mice that make human antibodies.

Production of Antibodies and Antibody-Producing Cell Lines

After immunization of an animal with a M-CSF antigen, antibodies and/orantibody-producing cells can be obtained from the animal. In someembodiments, anti-M-CSF antibody-containing serum is obtained from theanimal by bleeding or sacrificing the animal. The serum may be used asit is obtained from the animal, an immunoglobulin fraction may beobtained from the serum, or the anti-M-CSF antibodies may be purifiedfrom the serum.

In some embodiments, antibody-producing immortalized cell lines areprepared from cells isolated from the immunized animal. Afterimmunization, the animal is sacrificed and lymph node and/or splenic Bcells are immortalized. Methods of immortalizing cells include, but arenot limited to, transfecting them with oncogenes, infecting them with anoncogenic virus, cultivating them under conditions that select forimmortalized cells, subjecting them to carcinogenic or mutatingcompounds, fusing them with an immortalized cell, e.g., a myeloma cell,and inactivating a tumor suppressor gene. See, e.g., Harlow and Lane,supra. If fusion with myeloma cells is used, the myeloma cellspreferably do not secrete immunoglobulin polypeptides (a non-secretorycell line). Immortalized cells are screened using M-CSF, a portionthereof, or a cell expressing M-CSF. In a preferred embodiment, theinitial screening is performed using an enzyme-linked immunoassay(ELISA) or a radioimmunoassay. An example of ELISA screening is providedin WO 00/37504, incorporated herein by reference.

Anti-M-CSF antibody-producing cells, e.g., hybridomas, are selected,cloned and further screened for desirable characteristics, includingrobust growth, high antibody production and desirable antibodycharacteristics, as discussed further below. Hybridomas can be expandedin vivo in syngeneic animals, in animals that lack an immune system,e.g., nude mice, or in cell culture in vitro. Methods of selecting,cloning and expanding hybridomas are well known to those of ordinaryskill in the art.

In a preferred embodiment, the immunized animal is a non-human animalthat expresses human immunoglobulin genes and the splenic B cells arefused to a myeloma cell line from the same species as the non-humananimal. In a more preferred embodiment, the immunized animal is aXENOMOUSE® transgenic mouse that makes human antibodies and the myelomacell line is a non-secretory mouse myeloma. In an even more preferredembodiment, the myeloma cell line is P3-X63-AG8-653. See, e.g., ExampleI.

Thus, in one embodiment, the invention provides methods of producing acell line that produces a human monoclonal antibody or a fragmentthereof directed to M-CSF comprising (a) immunizing a non-humantransgenic animal described herein with M-CSF, a portion of M-CSF or acell or tissue expressing M-CSF; (b) allowing the transgenic animal tomount an immune response to M-CSF; (c) isolating B lymphocytes from atransgenic animal; (d) immortalizing the B lymphocytes; (e) creatingindividual monoclonal populations of the immortalized B lymphocytes; and(f) screening the immortalized B lymphocytes to identify an antibodydirected to M-CSF.

In another aspect, the invention provides hybridomas that produce anhuman anti-M-CSF antibody. In a preferred embodiment, the hybridomas aremouse hybridomas, as described above. In other embodiments, thehybridomas are produced in a non-human, non-mouse species such as rats,sheep, pigs, goats, cattle or horses. In another embodiment, thehybridomas are human hybridomas.

In another preferred embodiment, a transgenic animal is immunized withM-CSF, primary cells, e.g., spleen or peripheral blood cells, areisolated from an immunized transgenic animal and individual cellsproducing antibodies specific for the desired antigen are identified.Polyadenylated mRNA from each individual cell is isolated and reversetranscription polymerase chain reaction (RT-PCR) is performed usingsense primers that anneal to variable region sequences, e.g., degenerateprimers that recognize most or all of the FR1 regions of human heavy andlight chain variable region genes and antisense primers that anneal toconstant or joining region sequences. cDNAs of the heavy and light chainvariable regions are then cloned and expressed in any suitable hostcell, e.g., a myeloma cell, as chimeric antibodies with respectiveimmunoglobulin constant regions, such as the heavy chain and κ or λ,constant domains. See Babcook, J. S. et al., Proc. Natl. Acad. Sci. USA93:7843-48, 1996, herein incorporated by reference. Anti M-CSFantibodies may then be identified and isolated as described herein.

In another embodiment, phage display techniques can be used to providelibraries containing a repertoire of antibodies with varying affinitiesfor M-CSF. For production of such repertoires, it is unnecessary toimmortalize the B cells from the immunized animal. Rather, the primary Bcells can be used directly as a source of DNA. The mixture of cDNAsobtained from B cell, e.g., derived from spleens, is used to prepare anexpression library, for example, a phage display library transfectedinto E. coli. The resulting cells are tested for immunoreactivity toM-CSF. Techniques for the identification of high affinity humanantibodies from such libraries are described by Griffiths et al., EMBOJ., 13:3245-3260 (1994); Nissim et al., ibid, pp. 692-698 and byGriffiths et al., ibid, 12:725-734. Ultimately, clones from the libraryare identified which produce binding affinities of a desired magnitudefor the antigen and the DNA encoding the product responsible for suchbinding is recovered and manipulated for standard recombinantexpression. Phage display libraries may also be constructed usingpreviously manipulated nucleotide sequences and screened in a similarfashion. In general, the cDNAs encoding heavy and light chains areindependently supplied or linked to form Fv analogs for production inthe phage library.

The phage library is then screened for the antibodies with the highestaffinities for M-CSF and the genetic material recovered from theappropriate clone. Further rounds of screening can increase affinity ofthe original antibody isolated.

In another aspect, the invention provides hybridomas that produce anhuman anti-M-CSF antibody. In a preferred embodiment, the hybridomas aremouse hybridomas, as described above. In other embodiments, thehybridomas are produced in a non-human, non-mouse species such as rats,sheep, pigs, goats, cattle or horses. In another embodiment, thehybridomas are human hybridomas.

Nucleic Acids, Vectors, Host Cells, and Recombinant Methods of MakingAntibodies

Nucleic Acids

The present invention also encompasses nucleic acid molecules encodinganti-M-CSF antibodies. In some embodiments, different nucleic acidmolecules encode a heavy chain and a light chain of an anti-M-CSFimmunoglobulin. In other embodiments, the same nucleic acid moleculeencodes a heavy chain an a light chain of an anti-M-CSF immunoglobulin.In one embodiment, the nucleic acid encodes a M-CSF antibody of theinvention.

In some embodiments, the nucleic acid molecule encoding the variabledomain of the light chain comprises a human V_(κ)L5, O12, L2, B3, A27gene and a Jκ1, Jκ2, Jκ3, or Jκ4 gene.

In some embodiments, the nucleic acid molecule encoding the light chain,encodes an amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 mutations from the germline amino acid sequence. In some embodiments,the nucleic acid molecule comprises a nucleotide sequence that encodes aV_(L) amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10non-conservative amino acid substitutions and/or 1, 2, or 3non-conservative substitutions compared to germline sequence.Substitutions may be in the CDR regions, the framework regions, or inthe constant domain.

In some embodiments, the nucleic acid molecule encodes a V_(L) aminoacid sequence comprising one or more variants compared to germlinesequence that are identical to the variations found in the V_(L) of oneof the antibodies 252, 88, 100, 3.8.3, 2.7.3, 1.120.1, 9.14.4I, 8.10.3F,9.7.2IF, 9.14.4, 8.10.3, 9.7.2, 9.7.2C-Ser, 9.14.4C-Ser, 8.10.3C-Ser,8.10.3-CG2, 9.7.2-CG2, 9.7.2-CG4, 9.14.4-CG2, 9.14.4-CG4, 9.14.4-Ser,9.7.2-Ser, 8.10.3-Ser, 8.10.3-CG4, 8.10.3FG1 or 9.14.4G1.

In some embodiments, the nucleic acid molecule encodes at least threeamino acid mutations compared to the germline sequence found in theV_(L) of one of the antibodies 252, 88, 100, 3.8.3, 2.7.3, 1.120.1,9.14.4, 8.10.3, or 9.7.2.

In some embodiments, the nucleic acid molecule comprises a nucleotidesequence that encodes the V_(L) amino acid sequence of monoclonalantibody 252 (SEQ ID NO: 4), 88 (SEQ ID NO: 8), 100 (SEQ ID NO: 12),3.8.3 (SEQ ID NO: 16), 2.7.3 (SEQ ID NO: 20), 1.120.1 (SEQ ID NO: 24),9.14.4I (SEQ ID NO: 28), 8.10.3F (SEQ ID NO: 32), 9.7.2IF (SEQ ID NO:36), 9.14.4 (SEQ ID NO: 28), 8.10.3 (SEQ ID NO: 44), 9.7.2 (SEQ ID NO:48), 9.7.2C-Ser (SEQ ID NO: 52), 9.14.4C-Ser (SEQ ID NO: 56),8.10.3C-Ser (SEQ ID NO: 60), 8.10.3-CG2 (SEQ ID NO: 60), 9.7.2-CG2 (SEQID NO: 52), 9.7.2-CG4 (SEQ ID NO: 52), 9.14.4-CG2 (SEQ ID NO: 56),9.14.4-CG4 (SEQ ID NO: 56), 9.14.4-Ser (SEQ ID NO: 28), 9.7.2-Ser (SEQID NO: 48), 8.10.3-Ser (SEQ ID NO: 44), 8.10.3-CG4 (SEQ ID NO: 60)8.10.3FG1 (SEQ ID NO: 32) or 9.14.4G1 (SEQ ID NO: 28), or a portionthereof. In some embodiments, said portion comprises at least the CDR2region. In some embodiments, the nucleic acid encodes the amino acidsequence of the light chain CDRs of said antibody. In some embodiments,said portion is a contiguous portion comprising CDR1-CDR3.

In some embodiments, the nucleic acid molecule comprises a nucleotidesequence that encodes the light chain amino acid sequence of one of SEQID NOS: 4, 8, 12, 16, 20, 24, 28, 32, 36, 44, 48, 52, 56 or 60. In somepreferred embodiments, the nucleic acid molecule comprises the lightchain nucleotide sequence of SEQ ID NOS: 3, 7, 11, 27, 31, 35, 43 or 47,or a portion thereof.

In some embodiments, the nucleic acid molecule encodes a V_(L) aminoacid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or99% identical to a V_(L) amino acid sequence shown in FIG. 1 or to aV_(L) amino acid sequences of any one of antibodies 252, 88, 100, 3.8.3,2.7.3, 1.120.1, 9.14.4I, 8.10.3F, 9.7.2IF, 9.14.4, 8.10.3, 9.7.2,9.7.2C-Ser, 9.14.4C-Ser, 8.10.3C-Ser, 8.10.3-CG2, 9.7.2-CG2, 9.7.2-CG4,9.14.4-CG2, 9.14.4-CG4, 9.14.4-Ser, 9.7.2-Ser, 8.10.3-Ser, 8.10.3-CG4,8.10.3FG1 or 9.14.4G1, or an amino acid sequence of any one of SEQ IDNOS: 4, 8, 12, 16, 20, 24, 28, 32, 36, 44, 48, 52, 56 or 60. Nucleicacid molecules of the invention include nucleic acids that hybridizeunder highly stringent conditions, such as those described above, to anucleic acid sequence encoding the light chain amino acid sequence ofSEQ ID NOS: 4, 8, 12, 16, 20, 24, 28, 32, 36, 44, 48, 52, 56 or 60, orthat has the light chain nucleic acid sequence of SEQ ID NOS: 3, 7, 11,27, 31, 35, 43 or 47.

In another embodiment, the nucleic acid encodes a full-length lightchain of an antibody selected from 252, 88, 100, 3.8.3, 2.7.3, 1.120.1,9.14.4I, 8.10.3F, 9.7.2IF, 9.14.4, 8.10.3, 9.7.2, 9.7.2C-Ser,9.14.4C-Ser, 8.10.3C-Ser, 8.10.3-CG2, 9.7.2-CG2, 9.7.2-CG4, 9.14.4-CG2,9.14.4-CG4, 9.14.4-Ser, 9.7.2-Ser, 8.10.3-Ser, 8.10.3-CG4, 8.10.3FG1 or9.14.4G1, or a light chain comprising the amino acid sequence of SEQ IDNOS: 4, 8, 12, 16, 20, 24, 28, 32, 36, 44, 48, 52, 56 or 60 and aconstant region of a light chain, or a light chain comprising amutation. Further, the nucleic acid may comprise the light chainnucleotide sequence of SEQ ID NOS: 3, 7, 11, 27, 31, 35, 43 or 47 andthe nucleotide sequence encoding a constant region of a light chain, ora nucleic acid molecule encoding a light chain comprise a mutation.

In another preferred embodiment, the nucleic acid molecule encodes thevariable domain of the heavy chain (V_(H)) that comprises a human V_(H)1-18, 3-33, 3-11, 3-23, 3-48, or 3-7 gene sequence or a sequence derivedtherefrom. In various embodiments, the nucleic acid molecule comprises ahuman V_(H) 1-18 gene, a D_(H)4-23 gene and a human J_(H)4 gene; a humanV_(H) 3-33 gene, a human D_(H)1-26 gene and a human J_(H)4 gene; a humanV_(H) 3-11 gene, a human D_(H)7-27 gene and a human J_(H)4 gene; a humanV_(H) 3-11 gene, a human D_(H) 7-27 gene and a human J_(H)6 gene; ahuman V_(H) 3-23 gene, a human D_(H)1-26 gene and a human J_(H)4 gene; ahuman V_(H) 3-7 gene, a human D_(H)6-13 gene and a human J_(H)4 gene; ahuman V_(H)3-11 gene, a human D_(H)7-27 gene, and a human J_(H)4b gene;a human V_(H)3-48 gene, a human D_(H)1-26 gene, and a human J_(H)4bgene; a human V_(H)3-11 gene, a human D_(H)6-13 gene, and a humanJ_(H)6b gene, or a sequence derived from the human genes.

In some embodiments, the nucleic acid molecule encodes an amino acidsequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17 or 18 mutations compared to the germline amino acid sequence ofthe human V, D or J genes. In some embodiments, said mutations are inthe V_(H) region. In some embodiments, said mutations are in the CDRregions.

In some embodiments, the nucleic acid molecule encodes one or more aminoacid mutations compared to the germline sequence that are identical toamino acid mutations found in the V_(H) of monoclonal antibody 252, 88,100, 3.8.3, 2.7.3, 1.120.1, 9.14.4I, 8.10.3F, 9.7.2IF, 9.14.4, 8.10.3,9.7.2, 9.7.2C-Ser, 9.14.4C-Ser, 8.10.3C-Ser, 8.10.3-CG2, 9.7.2-CG2,9.7.2-CG4, 9.14.4-CG2, 9.14.4-CG4, 9.14.4-Ser, 9.7.2-Ser, 8.10.3-Ser,8.10.3-CG4, 8.10.3FG1 or 9.14.4G1. In some embodiments, the nucleic acidencodes at least three amino acid mutations compared to the germlinesequences that are identical to at least three amino acid mutationsfound in one of the above-listed monoclonal antibodies.

In some embodiments, the nucleic acid molecule comprises a nucleotidesequence that encodes at least a portion of the V_(H) amino acidsequence of antibody 252 (SEQ ID NO: 2), 88 (SEQ ID NO: 6), 100 (SEQ IDNO: 10), 3.8.3 (SEQ ID NO: 14), 2.7.3 (SEQ ID NO: 18), 1.120.1 (SEQ IDNO: 22), 9.14.4I (SEQ ID NO: 26), 8.10.3F (SEQ ID NO: 30), 9.7.2IF (SEQID NO: 34), 9.14.4 (SEQ ID NO: 38), 8.10.3 (SEQ ID NO: 30), 9.7.2 (SEQID NO: 46), 9.7.2C-Ser (SEQ ID NO: 50), 9.14.4C-Ser (SEQ ID NO: 54),8.10.3C-Ser (SEQ ID NO: 58), 8.10.3-CG2 (SEQ ID NO: 62), 9.7.2-CG2 (SEQID NO: 66), 9.7.2-CG4 (SEQ ID NO: 70), 9.14.4-CG2 (SEQ ID NO: 74),9.14.4-CG4 (SEQ ID NO: 78), 9.14.4-Ser (SEQ ID NO: 82), 9.7.2-Ser (SEQID NO: 86), 8.10.3-Ser (SEQ ID NO: 90), 8.10.3-CG4 (SEQ ID NO: 94),8.10.3FG1 (SEQ ID NO: 98) or 9.14.4-G1 (SEQ ID NO: 102), or saidsequence having conservative amino acid mutations and/or a total ofthree or fewer non-conservative amino acid substitutions. In variousembodiments the sequence encodes one or more CDR regions, preferably aCDR3 region, all three CDR regions, a contiguous portion includingCDR1-CDR3, or the entire V_(H) region.

In some embodiments, the nucleic acid molecule comprises a heavy chainnucleotide sequence that encodes the amino acid sequence of one of SEQID NOS: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 46, 50, 54, 58, 62, 66,70, 74, 78, 82, 86, 90, 94, 98 or 102. In some preferred embodiments,the nucleic acid molecule comprises at least a portion of the heavychain nucleotide sequence of SEQ ID NO: 1, 5, 9, 25, 29, 33, 37, 45, 97or 101. In some embodiments, said portion encodes the V_(H) region, aCDR3 region, all three CDR regions, or a contiguous region includingCDR1-CDR3.

In some embodiments, the nucleic acid molecule encodes a V_(H) aminoacid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or99% identical to the V_(H) amino acid sequences shown in FIGS. 4A-4ZZ orto a V_(H) amino acid sequence of any one of SEQ ID NOS: 2, 6, 10, 14,18, 22, 26, 30, 34, 38, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90,94, 98 or 102. Nucleic acid molecules of the invention include nucleicacids that hybridize under highly stringent conditions, such as thosedescribed above, to a nucleotide sequence encoding the heavy chain aminoacid sequence of SEQ ID NOS: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 46,50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98 or 102 or that hasthe nucleotide sequence of SEQ ID NOS: 1, 5, 9, 25, 29, 33, 37, 45, 97or 101.

In another embodiment, the nucleic acid encodes a full-length heavychain of an antibody selected from 252, 88, 100, 3.8.3, 2.7.3, 1.120.1,9.14.4I, 8.10.3F, 9.7.2IF, 9.14.4, 8.10.3, 9.7.2, 9.7.2C-Ser,9.14.4C-Ser, 8.10.3C-Ser, 8.10.3-CG2, 9.7.2-CG2, 9.7.2-CG4, 9.14.4-CG2,9.14.4-CG4, 9.14.4-Ser, 9.7.2-Ser, 8.10.3-Ser, 8.10.3-CG4, 8.10.3FG1 or9.14.4G1, or a heavy chain having the amino acid sequence of SEQ ID NOS:2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 46, 50, 54, 58, 62, 66, 70, 74,78, 82, 86, 90, 94, 98 or 102 and a constant region of a heavy chain, ora heavy chain comprising a mutation. Further, the nucleic acid maycomprise the heavy chain nucleotide sequence of SEQ ID NOS: 1, 5, 9, 25,29, 33, 37, 45, 97 or 101 and a nucleotide sequence encoding a constantregion of a light chain, or a nucleic acid molecule encoding a heavychain comprising a mutation.

A nucleic acid molecule encoding the heavy or entire light chain of ananti-M-CSF antibody or portions thereof can be isolated from any sourcethat produces such antibody. In various embodiments, the nucleic acidmolecules are isolated from a B cell isolated from an animal immunizedwith M-CSF or from an immortalized cell derived from such a B cell thatexpresses an anti-M-CSF antibody. Methods of isolating mRNA encoding anantibody are well-known in the art. See, e.g., Sambrook et al. The mRNAmay be used to produce cDNA for use in the polymerase chain reaction(PCR) or cDNA cloning of antibody genes. In a preferred embodiment, thenucleic acid molecule is isolated from a hybridoma that has as one ofits fusion partners a human immunoglobulin-producing cell from anon-human transgenic animal. In an even more preferred embodiment, thehuman immunoglobulin producing cell is isolated from a XENOMOUSE®transgenic mouse that makes human antibodies. In another embodiment, thehuman immunoglobulin-producing cell is from a non-human, non-mousetransgenic animal, as described above. In another embodiment, thenucleic acid is isolated from a non-human, non-transgenic animal. Thenucleic acid molecules isolated from a non-human, non-transgenic animalmay be used, e.g., for humanized antibodies.

In some embodiments, a nucleic acid encoding a heavy chain of ananti-M-CSF antibody of the invention can comprise a nucleotide sequenceencoding a V_(H) domain of the invention joined in-frame to a nucleotidesequence encoding a heavy chain constant domain from any source.Similarly, a nucleic acid molecule encoding a light chain of ananti-M-CSF antibody of the invention can comprise a nucleotide sequenceencoding a V_(L) domain of the invention joined in-frame to a nucleotidesequence encoding a light chain constant domain from any source.

In a further aspect of the invention, nucleic acid molecules encodingthe variable domain of the heavy (V_(H)) and light (V_(L)) chains are“converted” to full-length antibody genes. In one embodiment, nucleicacid molecules encoding the V_(H) or V_(L) domains are converted tofull-length antibody genes by insertion into an expression vectoralready encoding heavy chain constant (C_(H)) or light chain (C_(L))constant domains, respectively, such that the V_(H) segment isoperatively linked to the C_(H) segment(s) within the vector, and theV_(L) segment is operatively linked to the C_(L) segment within thevector. In another embodiment, nucleic acid molecules encoding the V_(H)and/or V_(L) domains are converted into full-length antibody genes bylinking, e.g., ligating, a nucleic acid molecule encoding a V_(H) and/orV_(L) domains to a nucleic acid molecule encoding a C_(H) and/or C_(L)domain using standard molecular biological techniques. Nucleic acidsequences of human heavy and light chain immunoglobulin constant domaingenes are known in the art. See, e.g., Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed., NIH Publ. No. 91-3242,1991. Nucleic acid molecules encoding the full-length heavy and/or lightchains may then be expressed from a cell into which they have beenintroduced and the anti-M-CSF antibody isolated.

The nucleic acid molecules may be used to recombinantly express largequantities of anti-M-CSF antibodies. The nucleic acid molecules also maybe used to produce chimeric antibodies, bispecific antibodies, singlechain antibodies, immunoadhesins, diabodies, mutated antibodies andantibody derivatives, as described further below. If the nucleic acidmolecules are derived from a non-human, non-transgenic animal, thenucleic acid molecules may be used for antibody humanization, also asdescribed below.

In another embodiment, a nucleic acid molecule of the invention is usedas a probe or PCR primer for a specific antibody sequence. For instance,the nucleic acid can be used as a probe in diagnostic methods or as aPCR primer to amplify regions of DNA that could be used, inter alia, toisolate additional nucleic acid molecules encoding variable domains ofanti-M-CSF antibodies. In some embodiments, the nucleic acid moleculesare oligonucleotides. In some embodiments, the oligonucleotides are fromhighly variable regions of the heavy and light chains of the antibody ofinterest. In some embodiments, the oligonucleotides encode all or a partof one or more of the CDRs of antibody 252, 88, 100, 3.8.3, 2.7.3, or1.120.1, or variants thereof described herein.

Vectors

The invention provides vectors comprising nucleic acid molecules thatencode the heavy chain of an anti-M-CSF antibody of the invention or anantigen-binding portion thereof. The invention also provides vectorscomprising nucleic acid molecules that encode the light chain of suchantibodies or antigen-binding portion thereof. The invention furtherprovides vectors comprising nucleic acid molecules encoding fusionproteins, modified antibodies, antibody fragments, and probes thereof.

In some embodiments, the anti-M-CSF antibodies, or antigen-bindingportions of the invention are expressed by inserting DNAs encodingpartial or full-length light and heavy chains, obtained as describedabove, into expression vectors such that the genes are operativelylinked to necessary expression control sequences such as transcriptionaland transnational control sequences. Expression vectors includeplasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV),plant viruses such as cauliflower mosaic virus, tobacco mosaic virus,cosmids, YACs, EBV derived episomes, and the like. The antibody gene isligated into a vector such that transcriptional and transnationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene. Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used. The antibody light chaingene and the antibody heavy chain gene can be inserted into separatevectors. In a preferred embodiment, both genes are inserted into thesame expression vector. The antibody genes are inserted into theexpression vector by standard methods (e.g., ligation of complementaryrestriction sites on the antibody gene fragment and vector, or blunt endligation if no restriction sites are present).

A convenient vector is one that encodes a functionally complete humanC_(H) or C_(L) immunoglobulin sequence, with appropriate restrictionsites engineered so that any V_(H) or V_(L) sequence can easily beinserted and expressed, as described above. In such vectors, splicingusually occurs between the splice donor site in the inserted J regionand the splice acceptor site preceding the human C domain, and also atthe splice regions that occur within the human C_(H) exons.Polyadenylation and transcription termination occur at nativechromosomal sites downstream of the coding regions. The recombinantexpression vector also can encode a signal peptide that facilitatessecretion of the antibody chain from a host cell. The antibody chaingene may be cloned into the vector such that the signal peptide islinked in-frame to the amino terminus of the immunoglobulin chain. Thesignal peptide can be an immunoglobulin signal peptide or a heterologoussignal peptide (i.e., a signal peptide from a non-immunoglobulinprotein).

In addition to the antibody chain genes, the recombinant expressionvectors of the invention carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. It will beappreciated by those skilled in the art that the design of theexpression vector, including the selection of regulatory sequences maydepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, etc. Preferred regulatorysequences for mammalian host cell expression include viral elements thatdirect high levels of protein expression in mammalian cells, such aspromoters and/or enhancers derived from retroviral LTRs, cytomegalovirus(CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (suchas the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus majorlate promoter (AdMLP)), polyoma and strong mammalian promoters such asnative immunoglobulin and actin promoters. For further description ofviral regulatory elements, and sequences thereof, see e.g., U.S. Pat.No. 5,168,062, U.S. Pat. No. 4,510,245 and U.S. Pat. No. 4,968,615.Methods for expressing antibodies in plants, including a description ofpromoters and vectors, as well as transformation of plants is known inthe art. See, e.g., U.S. Pat. No. 6,517,529, herein incorporated byreference. Methods of expressing polypeptides in bacterial cells orfungal cells, e.g., yeast cells, are also well known in the art.

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017). For example, typically theselectable marker gene confers resistance to drugs, such as G418,hygromycin or methotrexate, on a host cell into which the vector hasbeen introduced. Preferred selectable marker genes include thedihydrofolate reductase (DHFR) gene (for use in dhfr-host cells withmethotrexate selection/amplification), the neomycin resistance gene (forG418 selection), and the glutamate synthetase gene.

Non-Hybridoma Host Cells and Methods of Recombinantly Producing Protein

Nucleic acid molecules encoding anti-M-CSF antibodies and vectorscomprising these nucleic acid molecules can be used for transfection ofa suitable mammalian, plant, bacterial or yeast host cell.Transformation can be by any known method for introducingpolynucleotides into a host cell. Methods for introduction ofheterologous polynucleotides into mammalian cells are well known in theart and include dextran-mediated transfection, calcium phosphateprecipitation, polybrene-mediated transfection, protoplast fusion,electroporation, encapsulation of the polynucleotide(s) in liposomes,and direct microinjection of the DNA into nuclei. In addition, nucleicacid molecules may be introduced into mammalian cells by viral vectors.Methods of transforming cells are well known in the art. See, e.g., U.S.Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (which patentsare hereby incorporated herein by reference). Methods of transformingplant cells are well known in the art, including, e.g.,Agrobacterium-mediated transformation, biolistic transformation, directinjection, electroporation and viral transformation. Methods oftransforming bacterial and yeast cells are also well known in the art.

Mammalian cell lines available as hosts for expression are well known inthe art and include many immortalized cell lines available from theAmerican Type Culture Collection (ATCC). These include, inter alia,Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, babyhamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and a numberof other cell lines. Cell lines of particular preference are selectedthrough determining which cell lines have high expression levels. Othercell lines that may be used are insect cell lines, such as Sf9 cells.When recombinant expression vectors encoding antibody genes areintroduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or, more preferably,secretion of the antibody into the culture medium in which the hostcells are grown. Antibodies can be recovered from the culture mediumusing standard protein purification methods. Plant host cells include,e.g., Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, etc.Bacterial host cells include E. coli and Streptomyces species. Yeasthost cells include Schizosaccharomyces pombe, Saccharomyces cerevisiaeand Pichia pastoris.

Further, expression of antibodies of the invention (or other moietiestherefrom) from production cell lines can be enhanced using a number ofknown techniques. For example, the glutamine synthetase gene expressionsystem (the GS system) is a common approach for enhancing expressionunder certain conditions. The GS system is discussed in whole or part inconnection with European Patent Nos. 0 216 846, 0 256 055, and 0 323 997and European Patent Application No. 89303964.4.

It is possible that antibodies expressed by different cell lines or intransgenic animals will have different glycosylation from each other.However, all antibodies encoded by the nucleic acid molecules providedherein, or comprising the amino acid sequences provided herein are partof the instant invention, regardless of the glycosylation state orpattern or modification of the antibodies.

Transgenic Animals and Plants

Anti-M-CSF antibodies of the invention also can be producedtransgenically through the generation of a mammal or plant that istransgenic for the immunoglobulin heavy and light chain sequences ofinterest and production of the antibody in a recoverable form therefrom.In connection with the transgenic production in mammals, anti-M-CSFantibodies can be produced in, and recovered from, the milk of goats,cows, or other mammals. See, e.g., U.S. Pat. Nos. 5,827,690, 5,756,687,5,750,172, and 5,741,957. In some embodiments, non-human transgenicanimals that comprise human immunoglobulin loci are immunized with M-CSFor an immunogenic portion thereof, as described above. Methods formaking antibodies in plants, yeast or fungi/algae are described, e.g.,in U.S. Pat. No. 6,046,037 and U.S. Pat. No. 5,959,177.

In some embodiments, non-human transgenic animals or plants are producedby introducing one or more nucleic acid molecules encoding an anti-M-CSFantibody of the invention into the animal or plant by standardtransgenic techniques. See Hogan and U.S. Pat. No. 6,417,429, supra. Thetransgenic cells used for making the transgenic animal can be embryonicstem cells or somatic cells. The transgenic non-human organisms can bechimeric, nonchimeric heterozygotes, and nonchimeric homozygotes. See,e.g., Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual2ed., Cold Spring Harbor Press (1999); Jackson et al., Mouse Geneticsand Transgenics: A Practical Approach, Oxford University Press (2000);and Pinkert, Transgenic Animal Technology: A Laboratory Handbook,Academic Press (1999). In some embodiments, the transgenic non-humananimals have a targeted disruption and replacement by a targetingconstruct that encodes a heavy chain and/or a light chain of interest.In a preferred embodiment, the transgenic animals comprise and expressnucleic acid molecules encoding heavy and light chains that specificallybind to M-CSF, preferably human M-CSF. In some embodiments, thetransgenic animals comprise nucleic acid molecules encoding a modifiedantibody such as a single-chain antibody, a chimeric antibody or ahumanized antibody. The anti-M-CSF antibodies may be made in anytransgenic animal. In a preferred embodiment, the non-human animals aremice, rats, sheep, pigs, goats, cattle or horses. The non-humantransgenic animal expresses said encoded polypeptides in blood, milk,urine, saliva, tears, mucus and other bodily fluids.

Phage Display Libraries

The invention provides a method for producing an anti-M-CSF antibody orantigen-binding portion thereof comprising the steps of synthesizing alibrary of human antibodies on phage, screening the library with M-CSFor a portion thereof, isolating phage that bind M-CSF, and obtaining theantibody from the phage. By way of example, one method for preparing thelibrary of antibodies for use in phage display techniques comprises thesteps of immunizing a non-human animal comprising human immunoglobulinloci with M-CSF or an antigenic portion thereof to create an immuneresponse, extracting antibody producing cells from the immunized animal;isolating RNA from the extracted cells, reverse transcribing the RNA toproduce cDNA, amplifying the cDNA using a primer, and inserting the cDNAinto a phage display vector such that antibodies are expressed on thephage. Recombinant anti-M-CSF antibodies of the invention may beobtained in this way.

Recombinant anti-M-CSF human antibodies of the invention can be isolatedby screening a recombinant combinatorial antibody library. Preferablythe library is a scFv phage display library, generated using human V_(L)and V_(H) cDNAs prepared from mRNA isolated from B cells. Methodologiesfor preparing and screening such libraries are known in the art. Thereare commercially available kits for generating phage display libraries(e.g., the Pharmacia Recombinant Phage Antibody System, catalog no.27-9400-01; and the Stratagene SURFZAP® phage display kit, catalog no.240612). There also are other methods and reagents that can be used ingenerating and screening antibody display libraries (see, e.g., U.S.Pat. No. 5,223,409; PCT Publication Nos. WO 92/18619, WO 91/17271, WO92/20791, WO 92/15679, WO 93/01288, WO 92/01047, WO 92/09690; Fuchs etal., Bio/Technology 9:1370-1372 (1991); Hay et al., Hum. Antibod.Hybridomas 3:81-85 (1992); Huse et al., Science 246:1275-1281 (1989);McCafferty et al., Nature 348:552-554 (1990); Griffiths et al., EMBO J.12:725-734 (1993); Hawkins et al., J. Mol. Biol. 226:889-896 (1992);Clackson et al., Nature 352:624-628 (1991); Gram et al., Proc. Natl.Acad. Sci. USA 89:3576-3580 (1992); Garrad et al., Bio/Technology9:1373-1377 (1991); Hoogenboom et al., Nuc. Acid Res. 19:4133-4137(1991); and Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982(1991).

In one embodiment, to isolate human anti-M-CSF antibodies with thedesired characteristics, a human anti-M-CSF antibody as described hereinis first used to select human heavy and light chain sequences havingsimilar binding activity toward M-CSF, using the epitope imprintingmethods described in PCT Publication No. WO 93/06213. The antibodylibraries used in this method are preferably scFv libraries prepared andscreened as described in PCT Publication No. WO 92/01047, McCafferty etal., Nature 348:552-554 (1990); and Griffiths et al., EMBO J. 12:725-734(1993). The scFv antibody libraries preferably are screened using humanM-CSF as the antigen.

Once initial human V_(L) and V_(H) domains are selected, “mix and match”experiments are performed, in which different pairs of the initiallyselected V_(L) and V_(H) segments are screened for M-CSF binding toselect preferred V_(L)/V_(H) pair combinations. Additionally, to furtherimprove the quality of the antibody, the V_(L) and V_(H) segments of thepreferred V_(L)/V_(H) pair(s) can be randomly mutated, preferably withinthe CDR3 region of V_(H) and/or V_(L), in a process analogous to the invivo somatic mutation process responsible for affinity maturation ofantibodies during a natural immune response. This in vitro affinitymaturation can be accomplished by amplifying V_(H) and V_(L) domainsusing PCR primers complimentary to the V_(H) CDR3 or V_(L) CDR3,respectively, which primers have been “spiked” with a random mixture ofthe four nucleotide bases at certain positions such that the resultantPCR products encode V_(H) and V_(L) segments into which random mutationshave been introduced into the V_(H) and/or V_(L) CDR3 regions. Theserandomly mutated V_(H) and V_(L) segments can be re-screened for bindingto M-CSF.

Following screening and isolation of an anti-M-CSF antibody of theinvention from a recombinant immunoglobulin display library, nucleicacids encoding the selected antibody can be recovered from the displaypackage (e.g., from the phage genome) and subcloned into otherexpression vectors by standard recombinant DNA techniques. If desired,the nucleic acid can further be manipulated to create other antibodyforms of the invention, as described below. To express a recombinanthuman antibody isolated by screening of a combinatorial library, the DNAencoding the antibody is cloned into a recombinant expression vector andintroduced into a mammalian host cells, as described above.

Class Switching

Another aspect of the invention provides a method for converting theclass or subclass of an anti-M-CSF antibody to another class orsubclass. In some embodiments, a nucleic acid molecule encoding a V_(L)or V_(H) that does not include any nucleic acid sequences encoding C_(L)or C_(H) is isolated using methods well-known in the art. The nucleicacid molecule then is operatively linked to a nucleic acid sequenceencoding a C_(L) or C_(H) from a desired immunoglobulin class orsubclass. This can be achieved using a vector or nucleic acid moleculethat comprises a C_(L) or C_(H) chain, as described above. For example,an anti-M-CSF antibody that was originally IgM can be class switched toan IgG. Further, the class switching may be used to convert one IgGsubclass to another, e.g., from IgG1 to IgG2. Another method forproducing an antibody of the invention comprising a desired isotypecomprises the steps of isolating a nucleic acid encoding a heavy chainof an anti-M-CSF antibody and a nucleic acid encoding a light chain ofan anti-M-CSF antibody, isolating the sequence encoding the V_(H)region, ligating the V_(H) sequence to a sequence encoding a heavy chainconstant domain of the desired isotype, expressing the light chain geneand the heavy chain construct in a cell, and collecting the anti-M-CSFantibody with the desired isotype.

In some embodiments, anti-M-CSF antibodies of the invention have theserine at position 228 (according to the EU-numbering convention) of theheavy chain changed to a proline. Accordingly, the CPSC sub-sequence inthe F_(C) region of IgG4 becomes CPPC, which is the sub-sequence inIgG1. (Aalberse, R. C. and Schuurman, J., Immunology, 105:9-19 (2002)).For example, the serine at residue 243 SEQ ID NO: 46 (which correspondsto reside 228 in the EU-numbering convention) would become proline.Similarly, the serine at residue 242 of SEQ ID NO: 38 (which correspondsto reside 228 in the EU-numbering convention) would become proline. Insome embodiments, the framework region of the IgG4 antibody can beback-mutated to the germline framework sequence. Some embodimentscomprise both the back-mutates framework region and the serine toproline change in the F_(C) region. See, e.g., SEQ ID NO: 54 (antibody9.14.4C-Ser) and SEQ ID NO: 58 (antibody 8.10.3C-Ser) in Table 1.

Deimmunized Antibodies

Another way of producing antibodies with reduced immunogenicity is thedeimmunization of antibodies. In another aspect of the invention, theantibody may be deimmunized using the techniques described in, e.g., PCTPublication Nos. WO98/52976 and WO00/34317 (which incorporated herein byreference in their entirety).

Mutated Antibodies

In another embodiment, the nucleic acid molecules, vectors and hostcells may be used to make mutated anti-M-CSF antibodies. The antibodiesmay be mutated in the variable domains of the heavy and/or light chains,e.g., to alter a binding property of the antibody. For example, amutation may be made in one or more of the CDR regions to increase ordecrease the K_(D) of the antibody for M-CSF, to increase or decreasek_(off), or to alter the binding specificity of the antibody. Techniquesin site-directed mutagenesis are well-known in the art. See, e.g.,Sambrook et al. and Ausubel et al., supra. In a preferred embodiment,mutations are made at an amino acid residue that is known to be changedcompared to germline in a variable domain of an anti-M-CSF antibody. Inanother embodiment, one or more mutations are made at an amino acidresidue that is known to be changed compared to the germline in a CDRregion or framework region of a variable domain, or in a constant domainof a monoclonal antibody 252, 88, 100, 3.8.3, 2.7.3, 1.120.1, 9.14.4I,8.10.3F, 9.7.2IF, 9.14.4, 8.10.3, 9.7.2, 9.7.2C-Ser, 9.14.4C-Ser,8.10.3C-Ser, 8.10.3-CG2, 9.7.2-CG2, 9.7.2-CG4, 9.14.4-CG2, 9.14.4-CG4,9.14.4-Ser, 9.7.2-Ser, 8.10.3-Ser, 8.10.3-CG4, 8.10.3FG1 or 9.14.4G1. Inanother embodiment, one or more mutations are made at an amino acidresidue that is known to be changed compared to the germline in a CDRregion or framework region of a variable domain of a heavy chain aminoacid sequence selected from SEQ ID NOS: 2, 6, 10, 14, 18, 22, 26, 30,34, 38, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98 or 102,or whose heavy chain nucleotide sequence is presented in SEQ ID NOS: 1,5, 9, 25, 29, 33, 37, 45, 97 or 101. In another embodiment, one or moremutations are made at an amino acid residue that is known to be changedcompared to the germline in a CDR region or framework region of avariable domain of a light chain amino acid sequence selected from SEQID NOS: 4, 8, 12, 16, 20, 24, 28, 32, 36, 44, 48, 52, 56 or 60, or whoselight chain nucleotide sequence is presented in SEQ ID NOS: 3, 7, 11,27, 31, 35, 43 or 47.

In one embodiment, the framework region is mutated so that the resultingframework region(s) have the amino acid sequence of the correspondinggermline gene. A mutation may be made in a framework region or constantdomain to increase the half-life of the anti-M-CSF antibody. See, e.g.,PCT Publication No. WO 00/09560, herein incorporated by reference. Amutation in a framework region or constant domain also can be made toalter the immunogenicity of the antibody, to provide a site for covalentor non-covalent binding to another molecule, or to alter such propertiesas complement fixation, FcR binding and antibody-dependent cell-mediatedcytotoxicity (ADCC). According to the invention, a single antibody mayhave mutations in any one or more of the CDRs or framework regions ofthe variable domain or in the constant domain.

In some embodiments, there are from 1 to 8 including any number inbetween, amino acid mutations in either the V_(H) or V_(L) domains ofthe mutated anti-M-CSF antibody compared to the anti-M-CSF antibodyprior to mutation. In any of the above, the mutations may occur in oneor more CDR regions. Further, any of the mutations can be conservativeamino acid substitutions. In some embodiments, there are no more than 5,4, 3, 2, or 1 amino acid changes in the constant domains.

Modified Antibodies

In another embodiment, a fusion antibody or immunoadhesin may be madethat comprises all or a portion of an anti-M-CSF antibody of theinvention linked to another polypeptide. In a preferred embodiment, onlythe variable domains of the anti-M-CSF antibody are linked to thepolypeptide. In another preferred embodiment, the V_(H) domain of ananti-M-CSF antibody is linked to a first polypeptide, while the V_(L)domain of an anti-M-CSF antibody is linked to a second polypeptide thatassociates with the first polypeptide in a manner such that the V_(H)and V_(L) domains can interact with one another to form an antibodybinding site. In another preferred embodiment, the V_(H) domain isseparated from the V_(L) domain by a linker such that the V_(H) andV_(L) domains can interact with one another (see below under SingleChain Antibodies). The V_(H)-linker-V_(L) antibody is then linked to thepolypeptide of interest. The fusion antibody is useful for directing apolypeptide to a M-CSF-expressing cell or tissue. The polypeptide may bea therapeutic agent, such as a toxin, growth factor or other regulatoryprotein, or may be a diagnostic agent, such as an enzyme that may beeasily visualized, such as horseradish peroxidase. In addition, fusionantibodies can be created in which two (or more) single-chain antibodiesare linked to one another. This is useful if one wants to create adivalent or polyvalent antibody on a single polypeptide chain, or if onewants to create a bispecific antibody.

To create a single chain antibody, (scFv) the V_(H)- and V_(L)-encodingDNA fragments are operatively linked to another fragment encoding aflexible linker, e.g., encoding the amino acid sequence (Gly₄-Ser)₃,such that the V_(H) and V_(L) sequences can be expressed as a contiguoussingle-chain protein, with the V_(L) and V_(H) domains joined by theflexible linker. See, e.g., Bird et al., Science 242:423-426 (1988);Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988);McCafferty et al., Nature 348:552-554 (1990). The single chain antibodymay be monovalent, if only a single V_(H) and V_(L) are used, bivalent,if two V_(H) and V_(L) are used, or polyvalent, if more than two V_(H)and V_(L) are used. Bispecific or polyvalent antibodies may be generatedthat bind specifically to M-CSF and to another molecule.

In other embodiments, other modified antibodies may be prepared usinganti-M-CSF antibody-encoding nucleic acid molecules. For instance,“Kappa bodies” (Ill et al., Protein Eng. 10: 949-57 (1997)),“Minibodies” (Martin et al., EMBO J. 13: 5303-9 (1994)), “Diabodies”(Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993)), or“Janusins” (Traunecker et al., EMBO J. 10:3655-3659 (1991) andTraunecker et al., Int. J. Cancer (Suppl.) 7:51-52 (1992)) may beprepared using standard molecular biological techniques following theteachings of the specification.

Bispecific antibodies or antigen-binding fragments can be produced by avariety of methods including fusion of hybridomas or linking of Fab′fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990), Kostelny et al., J. Immunol. 148:1547-1553 (1992). Inaddition, bispecific antibodies may be formed as “diabodies” or“Janusins.” In some embodiments, the bispecific antibody binds to twodifferent epitopes of M-CSF. In some embodiments, the bispecificantibody has a first heavy chain and a first light chain from monoclonalantibody 252, 88, 100, 3.8.3, 2.7.3, 1.120.1, 9.14.4I, 8.10.3F, 9.7.2IF,9.14.4, 8.10.3, or 9.7.2 and an additional antibody heavy chain andlight chain. In some embodiments, the additional light chain and heavychain also are from one of the above-identified monoclonal antibodies,but are different from the first heavy and light chains.

In some embodiments, the modified antibodies described above areprepared using one or more of the variable domains or CDR regions from ahuman anti-M-CSF monoclonal antibody provided herein, from an amino acidsequence of said monoclonal antibody, or from a heavy chain or lightchain encoded by a nucleic acid sequence encoding said monoclonalantibody.

Derivatized and Labeled Antibodies

An anti-M-CSF antibody or antigen-binding portion of the invention canbe derivatized or linked to another molecule (e.g., another peptide orprotein). In general, the antibodies or portion thereof is derivatizedsuch that the M-CSF binding is not affected adversely by thederivatization or labeling. Accordingly, the antibodies and antibodyportions of the invention are intended to include both intact andmodified forms of the human anti-M-CSF antibodies described herein. Forexample, an antibody or antibody portion of the invention can befunctionally linked (by chemical coupling, genetic fusion, noncovalentassociation or otherwise) to one or more other molecular entities, suchas another antibody (e.g., a bispecific antibody or a diabody), adetection agent, a cytotoxic agent, a pharmaceutical agent, and/or aprotein or peptide that can mediate associate of the antibody orantibody portion with another molecule (such as a streptavidin coreregion or a polyhistidine tag).

One type of derivatized antibody is produced by crosslinking two or moreantibodies (of the same type or of different types, e.g., to createbispecific antibodies). Suitable crosslinkers include those that areheterobifunctional, having two distinctly reactive groups separated byan appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimideester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkersare available from Pierce Chemical Company, Rockford, Ill.

Another type of derivatized antibody is a labeled antibody. Usefuldetection agents with which an antibody or antigen-binding portion ofthe invention may be derivatized include fluorescent compounds,including fluorescein, fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanidephosphors and the like. An antibody can also be labeled with enzymesthat are useful for detection, such as horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase andthe like. When an antibody is labeled with a detectable enzyme, it isdetected by adding additional reagents that the enzyme uses to produce areaction product that can be discerned. For example, when the agenthorseradish peroxidase is present, the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. An antibody can also be labeled with biotin, and detectedthrough indirect measurement of avidin or streptavidin binding. Anantibody can also be labeled with a predetermined polypeptide epitoperecognized by a secondary reporter (e.g., leucine zipper pair sequences,binding sites for secondary antibodies, metal binding domains, epitopetags). In some embodiments, labels are attached by spacer arms ofvarious lengths to reduce potential steric hindrance.

An anti-M-CSF antibody can also be labeled with a radiolabeled aminoacid. The radiolabeled anti-M-CSF antibody can be used for bothdiagnostic and therapeutic purposes. For instance, the radiolabeledanti-M-CSF antibody can be used to detect M-CSF-expressing tumors byx-ray or other diagnostic techniques. Further, the radiolabeledanti-M-CSF antibody can be used therapeutically as a toxin for cancerouscells or tumors. Examples of labels for polypeptides include, but arenot limited to, the following radioisotopes or radionuclides—³H, ¹⁴C,¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, and ¹³¹I.

An anti-M-CSF antibody can also be derivatized with a chemical groupsuch as polyethylene glycol (PEG), a methyl or ethyl group, or acarbohydrate group. These groups are useful to improve the biologicalcharacteristics of the antibody, e.g., to increase serum half-life or toincrease tissue binding.

Pharmaceutical Compositions and Kits

The invention also relates to compositions comprising a human anti-M-CSFantagonist antibody for the treatment of subjects in need of treatmentfor rheumatoid arthritis, osteoporosis, or atherosclerosis. In someembodiments, the subject of treatment is a human. In other embodiments,the subject is a veterinary subject. Hyperproliferative disorders wheremonocytes play a role that may be treated by an antagonist anti-M-CSFantibody of the invention can involve any tissue or organ and includebut are not limited to brain, lung, squamous cell, bladder, gastric,pancreatic, breast, head, neck, liver, renal, ovarian, prostate,colorectal, esophageal, gynecological, nasopharynx, or thyroid cancers,melanomas, lymphomas, leukemias or multiple myelomas. In particular,human antagonist anti-M-CSF antibodies of the invention are useful totreat or prevent carcinomas of the breast, prostate, colon and lung.

This invention also encompasses compositions for the treatment of acondition selected from the group consisting of arthritis, psoriaticarthritis, Reiter's syndrome, gout, traumatic arthritis, rubellaarthritis and acute synovitis, rheumatoid arthritis, rheumatoidspondylitis, ankylosing spondylitis, osteoarthritis, gouty arthritis andother arthritic conditions, sepsis, septic shock, endotoxic shock, gramnegative sepsis, toxic shock syndrome, Alzheimer's disease, stroke,neurotrauma, asthma, adult respiratory distress syndrome, cerebralmalaria, chronic pulmonary inflammatory disease, silicosis, pulmonarysarcoidosis, bone resorption disease, osteoporosis, restenosis, cardiacand renal reperfusion injury, thrombosis, glomerularonephritis,diabetes, graft vs. host reaction, allograft rejection, inflammatorybowel disease, Crohn's disease, ulcerative colitis, multiple sclerosis,muscle degeneration, eczema, contact dermatitis, psoriasis, sunburn, orconjunctivitis shock in a mammal, including a human, comprising anamount of a human anti-M-CSF monoclonal antibody of the inventioneffective in such treatment and a pharmaceutically acceptable carrier.

Treatment may involve administration of one or more antagonistanti-M-CSF monoclonal antibodies of the invention, or antigen-bindingfragments thereof, alone or with a pharmaceutically acceptable carrier.As used herein, “pharmaceutically acceptable carrier” means any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Some examples of pharmaceutically acceptablecarriers are water, saline, phosphate buffered saline, dextrose,glycerol, ethanol and the like, as well as combinations thereof. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride inthe composition. Additional examples of pharmaceutically acceptablesubstances are wetting agents or minor amounts of auxiliary substancessuch as wetting or emulsifying agents, preservatives or buffers, whichenhance the shelf life or effectiveness of the antibody.

Anti-M-CSF antibodies of the invention and compositions comprising them,can be administered in combination with one or more other therapeutic,diagnostic or prophylactic agents. Additional therapeutic agents includeother anti-neoplastic, anti-tumor, anti-angiogenic or chemotherapeuticagents. Such additional agents may be included in the same compositionor administered separately. In some embodiments, one or more inhibitoryanti-M-CSF antibodies of the invention can be used as a vaccine or asadjuvants to a vaccine.

The compositions of this invention may be in a variety of forms, forexample, liquid, semi-solid and solid dosage forms, such as liquidsolutions (e.g., injectable and infusible solutions), dispersions orsuspensions, tablets, pills, powders, liposomes and suppositories. Thepreferred form depends on the intended mode of administration andtherapeutic application. Typical preferred compositions are in the formof injectable or infusible solutions, such as compositions similar tothose used for passive immunization of humans. The preferred mode ofadministration is parenteral (e.g., intravenous, subcutaneous,intraperitoneal, intramuscular). In a preferred embodiment, the antibodyis administered by intravenous infusion or injection. In anotherpreferred embodiment, the antibody is administered by intramuscular orsubcutaneous injection. In another embodiment, the invention includes amethod of treating a subject in need thereof with an antibody or anantigen-binding portion thereof that specifically binds to M-CSFcomprising the steps of: (a) administering an effective amount of anisolated nucleic acid molecule encoding the heavy chain or theantigen-binding portion thereof, an isolated nucleic acid moleculeencoding the light chain or the antigen-binding portion thereof, or boththe nucleic acid molecules encoding the light chain and the heavy chainor antigen-binding portions thereof; and (b) expressing the nucleic acidmolecule.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the anti-M-CSF antibody inthe required amount in an appropriate solvent with one or a combinationof ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The proper fluidity of a solution can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

The antibodies of the present invention can be administered by a varietyof methods known in the art, although for many therapeutic applications,the preferred route/mode of administration is subcutaneous,intramuscular, or intravenous infusion. As will be appreciated by theskilled artisan, the route and/or mode of administration will varydepending upon the desired results.

In certain embodiments, the antibody compositions active compound may beprepared with a carrier that will protect the antibody against rapidrelease, such as a controlled release formulation, including implants,transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems (J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978).

In certain embodiments, an anti-M-CSF antibody of the invention can beorally administered, for example, with an inert diluent or anassimilable edible carrier. The compound (and other ingredients, ifdesired) can also be enclosed in a hard or soft shell gelatin capsule,compressed into tablets, or incorporated directly into the subject'sdiet. For oral therapeutic administration, the anti-M-CSF antibodies canbe incorporated with excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. To administer a compound of the inventionby other than parenteral administration, it may be necessary to coat thecompound with, or co-administer the compound with, a material to preventits inactivation.

Additional active compounds also can be incorporated into thecompositions. In certain embodiments, an anti-M-CSF antibody of theinvention is co-formulated with and/or co-administered with one or moreadditional therapeutic agents. These agents include antibodies that bindother targets, antineoplastic agents, antitumor agents, chemotherapeuticagents, peptide analogues that inhibit M-CSF, soluble c-fms that canbind M-CSF, one or more chemical agents that inhibit M-CSF,anti-inflammatory agents, anti-coagulants, agents that lower bloodpressure (i.e, angiotensin-converting enzyme (ACE) inhibitors). Suchcombination therapies may require lower dosages of the anti-M-CSFantibody as well as the co-administered agents, thus avoiding possibletoxicities or complications associated with the various monotherapies.

Inhibitory anti-M-CSF antibodies of the invention and compositionscomprising them also may be administered in combination with othertherapeutic regimens, in particular in combination with radiationtreatment for cancer. The compounds of the present invention may also beused in combination with anticancer agents such as endostatin andangiostatin or cytotoxic drugs such as adriamycin, daunomycin,cis-platinum, etoposide, taxol, taxotere and alkaloids, such asvincristine, farnesyl transferase inhibitors, VEGF inhibitors, andantimetabolites such as methotrexate.

The compounds of the invention may also be used in combination withantiviral agents such as VIRACEPT® (nelfinavir mesylate), AZT, aciclovirand famciclovir, and antisepsis compounds such as Valant.

The compositions of the invention may include a “therapeuticallyeffective amount” or a “prophylactically effective amount” of anantibody or antigen-binding portion of the invention. A “therapeuticallyeffective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic result. Atherapeutically effective amount of the antibody or antibody portion mayvary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the antibody or antibodyportion to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the antibody or antibody portion are outweighedby the therapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typically,since a prophylactic dose is used in subjects prior to or at an earlierstage of disease, the prophylactically effective amount will be lessthan the therapeutically effective amount.

Dosage regimens can be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus can be administered, several divided doses can be administeredover time or the dose can be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the anti-M-CSF antibody or portion and the particulartherapeutic or prophylactic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an antibody for thetreatment of sensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody or antibody portion ofthe invention is 0.025 to 50 mg/kg, more preferably 0.1 to 50 mg/kg,more preferably 0.1-25, 0.1 to 10 or 0.1 to 3 mg/kg. It is to be notedthat dosage values may vary with the type and severity of the conditionto be alleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.

Another aspect of the present invention provides kits comprising ananti-M-CSF antibody or antigen-binding portion of the invention or acomposition comprising such an antibody or portion. A kit may include,in addition to the antibody or composition, diagnostic or therapeuticagents. A kit also can include instructions for use in a diagnostic ortherapeutic method. In a preferred embodiment, the kit includes theantibody or a composition comprising it and a diagnostic agent that canbe used in a method described below. In another preferred embodiment,the kit includes the antibody or a composition comprising it and one ormore therapeutic agents that can be used in a method described below.One embodiment of the invention is a kit comprising a container,instructions on the administration of an anti-M-CSF antibody to a humansuffering from an inflammatory disease, or instructions for measuringthe number of CD14+CD16+ monocytes in a biological sample and ananti-M-CSF antibody.

This invention also relates to compositions for inhibiting abnormal cellgrowth in a mammal comprising an amount of an antibody of the inventionin combination with an amount of a chemotherapeutic agent, wherein theamounts of the compound, salt, solvate, or prodrug, and of thechemotherapeutic agent are together effective in inhibiting abnormalcell growth. Many chemotherapeutic agents are known in the art. In someembodiments, the chemotherapeutic agent is selected from the groupconsisting of mitotic inhibitors, alkylating agents, anti-metabolites,intercalating antibiotics, growth factor inhibitors, cell cycleinhibitors, enzymes, topoisomerase inhibitors, biological responsemodifiers, anti-hormones, e.g. anti-androgens, and anti-angiogenesisagents.

Anti-angiogenic agents, such as MMP-2 (matrix-metalloproteinase 2)inhibitors, MMP-9 (matrix-metalloproteinase 9) inhibitors, and COX-II(cyclooxygenase II) inhibitors, can be used in conjunction with ananti-M-CSF antibody of the invention. Examples of useful COX-IIinhibitors include CELEBREX® (celecoxib), valdecoxib, and rofecoxib.Examples of useful matrix metalloproteinase inhibitors are described inWO 96/33172 (published Oct. 24, 1996), WO 96/27583 (published Mar. 7,1996), European Patent Application No. 97304971.1 (filed Jul. 8, 1997),European Patent Application No. 99308617.2 (filed Oct. 29, 1999), WO98/07697 (published Feb. 26, 1998), WO 98/03516 (published Jan. 29,1998), WO 98/34918 (published Aug. 13, 1998), WO 98/34915 (publishedAug. 13, 1998), WO 98/33768 (published Aug. 6, 1998), WO 98/30566(published Jul. 16, 1998), European Patent Publication 606,046(published Jul. 13, 1994), European Patent Publication 931,788(published Jul. 28, 1999), WO 90/05719 (published May 31, 1990), WO99/52910 (published Oct. 21, 1999), WO 99/52889 (published Oct. 21,1999), WO 99/29667 (published Jun. 17, 1999), PCT InternationalApplication No. PCT/IB98/01113 (filed Jul. 21, 1998), European PatentApplication No. 99302232.1 (filed Mar. 25, 1999), Great Britain patentapplication number 9912961.1 (filed Jun. 3, 1999), U.S. ProvisionalApplication No. 60/148,464 (filed Aug. 12, 1999), U.S. Pat. No.5,863,949 (issued Jan. 26, 1999), U.S. Pat. No. 5,861,510 (issued Jan.19, 1999), and European Patent Publication 780,386 (published Jun. 25,1997), all of which are incorporated herein in their entireties byreference. Preferred MMP inhibitors are those that do not demonstratearthralgia. More preferred, are those that selectively inhibit MMP-2and/or MMP-9 relative to the other matrix-metalloproteinases (i.e.MMP-1, MMP-3, MMP-4, MMP-5, MMP-6, MMP-7, MMP-8, MMP-10, MMP-11, MMP-12,and MMP-13). Some specific examples of MMP inhibitors useful in thepresent invention are AG-3340, RO 32-3555, RS 13-0830, and the compoundsrecited in the following list:3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-cyclopentyl)-amino]-propionicacid;3-exo-3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-8-oxa-bicyclo[3.2.1]octane-3-carboxylicacid hydroxyamide; (2R,3R)1-[4-(2-chloro-4-fluoro-benzyloxy)-benzenesulfonyl]-3-hydroxy-3-methyl-piperidine-2-carboxylicacid hydroxyamide;4-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-4-carboxylicacid hydroxyamide;3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-cyclobutyl)-amino]-propionicacid;4-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-4-carboxylicacid hydroxyamide; (R)3-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-3-carboxylicacid hydroxyamide; (2R,3R)1-[4-(4-fluoro-2-methyl-benzyloxy)-benzenesulfonyl]-3-hydroxy-3-methyl-piperidine-2-carboxylicacid hydroxyamide;3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-1-methyl-ethyl)-amino]-propionicacid;3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(4-hydroxycarbamoyl-tetrahydro-pyran-4-yl)-amino]-propionicacid;3-exo-3-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-8-oxa-bicyclo[3.2.1]octane-3-carboxylicacid hydroxyamide;3-endo-3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-8-oxa-bicyclo[3.2.1]octane-3-carboxylicacid hydroxyamide; and (R)3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-tetrahydro-furan-3-carboxylicacid hydroxyamide; and pharmaceutically acceptable salts and solvates ofsaid compounds.

A compound comprising a human anti-M-CSF monoclonal antibody of theinvention can also be used with signal transduction inhibitors, such asagents that can inhibit EGF-R (epidermal growth factor receptor)responses, such as EGF-R antibodies, EGF antibodies, and molecules thatare EGF-R inhibitors; VEGF (vascular endothelial growth factor)inhibitors, such as VEGF receptors and molecules that can inhibit VEGF;and erbB2 receptor inhibitors, such as organic molecules or antibodiesthat bind to the erbB2 receptor, for example, HERCEPTIN® (trastuzumab)(Genentech, Inc.). EGF-R inhibitors are described in, for example in WO95/19970 (published Jul. 27, 1995), WO 98/14451 (published Apr. 9,1998), WO 98/02434 (published Jan. 22, 1998), and U.S. Pat. No.5,747,498 (issued May 5, 1998), and such substances can be used in thepresent invention as described herein. EGFR-inhibiting agents include,but are not limited to, the monoclonal antibodies C225 and anti-EGFR22Mab (ImClone Systems Incorporated), ABX-EGF (Abgenix/Cell Genesys),EMD-7200 (Merck KgaA), EMD-5590 (Merck KgaA), MDX-447/H-477 (MedarexInc. and Merck KgaA), and the compounds ZD-1834, ZD-1838 and ZD-1839(AstraZeneca), PKI-166 (Novartis), PKI-166/CGP-75166 (Novartis), PTK 787(Novartis), CP 701 (Cephalon), leflunomide (Pharmacia/Sugen), CI-1033(Warner Lambert Parke Davis), CI-1033/PD 183,805 (Warner Lambert ParkeDavis), CL-387,785 (Wyeth-Ayerst), BBR-1611 (Boehringer MannheimGmbH/Roche), Naamidine A (Bristol Myers Squibb), RC-3940-II (Pharmacia),BIBX-1382 (Boehringer Ingelheim), OLX-103 (Merck & Co.), VRCTC-310(Ventech Research), EGF fusion toxin (Seragen Inc.), DAB-389(Seragen/Lilgand), ZM-252808 (Imperial Cancer Research Fund), RG-50864(INSERM), LFM-A12 (Parker Hughes Cancer Center), WHI-P97 (Parker HughesCancer Center), GW-282974 (Glaxo), KT-8391 (Kyowa Hakko) and EGF-RVaccine (York Medical/Centro de Immunologia Molecular (CIM)). These andother EGF-R-inhibiting agents can be used in the present invention.

VEGF inhibitors, for example SU-5416 and SU-6668 (Sugen Inc.), AVASTIN®(bevacizumab) (Genentech), SH-268 (Schering), and NX-1838 (NeXstar) canalso be combined with the compound of the present invention. VEGFinhibitors are described in, for example in WO 99/24440 (published May20, 1999), PCT International Application PCT/IB99/00797 (filed May 3,1999), in WO 95/21613 (published Aug. 17, 1995), WO 99/61422 (publishedDec. 2, 1999), U.S. Pat. No. 5,834,504 (issued Nov. 10, 1998), WO98/50356 (published Nov. 12, 1998), U.S. Pat. No. 5,883,113 (issued Mar.16, 1999), U.S. Pat. No. 5,886,020 (issued Mar. 23, 1999), U.S. Pat. No.5,792,783 (issued Aug. 11, 1998), WO 99/10349 (published Mar. 4, 1999),WO 97/32856 (published Sep. 12, 1997), WO 97/22596 (published Jun. 26,1997), WO 98/54093 (published Dec. 3, 1998), WO 98/02438 (published Jan.22, 1998), WO 99/16755 (published Apr. 8, 1999), and WO 98/02437(published Jan. 22, 1998), all of which are incorporated herein in theirentireties by reference. Other examples of some specific VEGF inhibitorsuseful in the present invention are IM862 (Cytran Inc.); anti-VEGFmonoclonal antibody of Genentech, Inc.; and angiozyme, a syntheticribozyme from Ribozyme and Chiron. These and other VEGF inhibitors canbe used in the present invention as described herein. ErbB2 receptorinhibitors, such as GW-282974 (Glaxo Wellcome plc), and the monoclonalantibodies AR-209 (Aronex Pharmaceuticals Inc.) and 2B-1 (Chiron), canfurthermore be combined with the compound of the invention, for examplethose indicated in WO 98/02434 (published Jan. 22, 1998), WO 99/35146(published Jul. 15, 1999), WO 99/35132 (published Jul. 15, 1999), WO98/02437 (published Jan. 22, 1998), WO 97/13760 (published Apr. 17,1997), WO 95/19970 (published Jul. 27, 1995), U.S. Pat. No. 5,587,458(issued Dec. 24, 1996), and U.S. Pat. No. 5,877,305 (issued Mar. 2,1999), which are all hereby incorporated herein in their entireties byreference. ErbB2 receptor inhibitors useful in the present invention arealso described in U.S. Pat. No. 6,465,449 (issued Oct. 15, 2002), and inU.S. Pat. No. 6,284,764 (issued Sep. 4, 2001), both of which areincorporated in their entireties herein by reference. The erbB2 receptorinhibitor compounds and substance described in the aforementioned PCTapplications, U.S. patents, and U.S. provisional applications, as wellas other compounds and substances that inhibit the erbB2 receptor, canbe used with the compound of the present invention in accordance withthe present invention.

Anti-survival agents include anti-IGF-IR antibodies and anti-integrinagents, such as anti-integrin antibodies.

Anti-inflammatory agents can be used in conjunction with an anti-M-CSFantibody of the invention. For the treatment of rheumatoid arthritis,the human anti-M-CSF antibodies of the invention may be combined withagents such as TNF-α inhibitors such as TNF drugs (such as REMICADE®(infliximab), CDP-870 and HUMIRA® (adalimumab)) and TNF receptorimmunoglobulin molecules (such as ENBREL® (etanercept)), IL-1inhibitors, receptor antagonists or soluble IL-1ra (e.g. KINERET®(anakinra) or ICE inhibitors), COX-2 inhibitors (such as celecoxib,rofecoxib, valdecoxib and etoricoxib), metalloprotease inhibitors(preferably MMP-13 selective inhibitors), p2X7 inhibitors, α2δ ligands(such as NEUROTIN® (gabapentin) and LYRICA® (pregabalin), low dosemethotrexate, leflunomide, hydroxychloroquine, d-penicillamine,auranofin or parenteral or oral gold. The compounds of the invention canalso be used in combination with existing therapeutic agents for thetreatment of osteoarthritis. Suitable agents to be used in combinationinclude standard non-steroidal anti-inflammatory agents (hereinafterNSAID's) such as piroxicam, diclofenac, propionic acids such asnaproxen, flurbiprofen, fenoprofen, ketoprofen and ibuprofen, fenamatessuch as mefenamic acid, indomethacin, sulindac, apazone, pyrazolonessuch as phenylbutazone, salicylates such as aspirin, COX-2 inhibitorssuch as celecoxib, valdecoxib, rofecoxib and etoricoxib, analgesics andintraarticular therapies such as corticosteroids and hyaluronic acidssuch as hyalgan and synvisc.

Anti-coagulant agents can be used in conjunction with an anti-M-CSFantibody of the invention. Examples of anti-coagulant agents include,but are not limited to, COUMADIN® (warfarin sodium), heparin, andLOVENOX® (enoxaparin sodium).

The human anti-M-CSF antibodies of the present invention may also beused in combination with cardiovascular agents such as calcium channelblockers, lipid lowering agents such as statins, fibrates,beta-blockers, Ace inhibitors, Angiotensin-2 receptor antagonists andplatelet aggregation inhibitors. The compounds of the present inventionmay also be used in combination with CNS agents such as antidepressants(such as sertraline), anti-Parkinsonian drugs (such as deprenyl, L-dopa,REQUIP® (ropinirole HCl), MIRAPEX® (pramipexole dihydrochloride), MAOBinhibitors such as selegine and rasagiline, comP inhibitors such asTasmar, A-2 inhibitors, dopamine reuptake inhibitors, NMDA antagonists,Nicotine agonists, Dopamine agonists and inhibitors of neuronal nitricoxide synthase), and anti-Alzheimer's drugs such as donepezil, tacrine,α2δ LIGANDS (such as NEUROTIN® (gabapentin) and LYRICA® (pregabalin))inhibitors, COX-2 inhibitors, propentofylline or metryfonate.

The human anti-M-CSF antibodies of the present invention may also beused in combination with osteoporosis agents such as roloxifene,droloxifene, lasofoxifene or fosomax and immunosuppressant agents suchas FK-506 and rapamycin.

Diagnostic Methods of Use

In another aspect, the invention provides diagnostic methods. Theanti-M-CSF antibodies can be used to detect M-CSF in a biological samplein vitro or in vivo. In one embodiment, the invention provides a methodfor diagnosing the presence or location of a M-CSF-expressing tumor in asubject in need thereof, comprising the steps of injecting the antibodyinto the subject, determining the expression of M-CSF in the subject bylocalizing where the antibody has bound, comparing the expression in thesubject with that of a normal reference subject or standard, anddiagnosing the presence or location of the tumor.

The anti-M-CSF antibodies can be used in a conventional immunoassay,including, without limitation, an ELISA, an RIA, FACS, tissueimmunohistochemistry, Western blot or immunoprecipitation. Theanti-M-CSF antibodies of the invention can be used to detect M-CSF fromhumans. In another embodiment, the anti-M-CSF antibodies can be used todetect M-CSF from primates such as cynomologus monkey, rhesus monkeys,chimpanzees or apes. The invention provides a method for detecting M-CSFin a biological sample comprising contacting a biological sample with ananti-M-CSF antibody of the invention and detecting the bound antibody.In one embodiment, the anti-M-CSF antibody is directly labeled with adetectable label. In another embodiment, the anti-M-CSF antibody (thefirst antibody) is unlabeled and a second antibody or other moleculethat can bind the anti-M-CSF antibody is labeled. As is well known toone of skill in the art, a second antibody is chosen that is able tospecifically bind the particular species and class of the firstantibody. For example, if the anti-M-CSF antibody is a human IgG, thenthe secondary antibody could be an anti-human-IgG. Other molecules thatcan bind to antibodies include, without limitation, Protein A andProtein G, both of which are available commercially, e.g., from PierceChemical Co.

Suitable labels for the antibody or secondary antibody have beendisclosed supra, and include various enzymes, prosthetic groups,fluorescent materials, luminescent materials and radioactive materials.Examples of suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; and examples ofsuitable radioactive material include ¹²⁵I, ¹³¹I, ¹³⁵S or ³H.

In other embodiments, M-CSF can be assayed in a biological sample by acompetition immunoassay utilizing M-CSF standards labeled with adetectable substance and an unlabeled anti-M-CSF antibody. In thisassay, the biological sample, the labeled M-CSF standards and theanti-M-CSF antibody are combined and the amount of labeled M-CSFstandard bound to the unlabeled antibody is determined. The amount ofM-CSF in the biological sample is inversely proportional to the amountof labeled M-CSF standard bound to the anti-M-CSF antibody.

One can use the immunoassays disclosed above for a number of purposes.For example, the anti-M-CSF antibodies can be used to detect M-CSF incells or on the surface of cells in cell culture, or secreted into thetissue culture medium. The anti-M-CSF antibodies can be used todetermine the amount of M-CSF on the surface of cells or secreted intothe tissue culture medium that have been treated with various compounds.This method can be used to identify compounds that are useful to inhibitor activate M-CSF expression or secretion. According to this method, onesample of cells is treated with a test compound for a period of timewhile another sample is left untreated. If the total level of M-CSF isto be measured, the cells are lysed and the total M-CSF level ismeasured using one of the immunoassays described above. The total levelof M-CSF in the treated versus the untreated cells is compared todetermine the effect of the test compound.

An immunoassay for measuring total M-CSF levels is an ELISA or Westernblot. If the cell surface level of M-CSF is to be measured, the cellsare not lysed, and the M-CSF cell surface levels can be measured usingone of the immunoassays described above. An immunoassay for determiningcell surface levels of M-CSF can include the steps of labeling the cellsurface proteins with a detectable label, such as biotin or ¹²⁵I,immunoprecipitating the M-CSF with an anti-M-CSF antibody and thendetecting the labeled M-CSF. Another immunoassay for determining thelocalization of M-CSF, e.g., cell surface levels, can beimmunohistochemistry. Methods such as ELISA, RIA, Western blot,immunohistochemistry, cell surface labeling of integral membraneproteins and immunoprecipitation are well known in the art. See, e.g.,Harlow and Lane, supra. In addition, the immunoassays can be scaled upfor high throughput screening in order to test a large number ofcompounds for inhibition or activation of M-CSF.

Another example of an immunoassay for measuring secreted M-CSF levelscan be an antigen capture assay, ELISA, immunohistochemistry assay,Western blot and the like using antibodies of the invention. If secretedM-CSF is to be measured, cell culture media or body fluid, such as bloodserum, urine, or synovial fluid, can be assayed for secreted M-CSFand/or cells can be lysed to release produced, but not yet secretedM-CSF. An immunoassay for determining secreted levels of M-CSF includesthe steps of labeling the secreted proteins with a detectable label,such as biotin or ¹²⁵I, immunoprecipitating the M-CSF with an anti-M-CSFantibody and then detecting the labeled M-CSF. Another immunoassay fordetermining secreted levels of M-CSF can include the steps of (a)pre-binding anti-M-CSF antibodies to the surface of a microtiter plate;(b) adding tissue culture cell media or body fluid containing thesecreted M-CSF to the wells of the microtiter plate to bind to theanti-M-CSF antibodies; (c) adding an antibody that will detect theanti-M-CSF antibody, e.g., anti-M-CSF labeled with digoxigenin thatbinds to an epitope of M-CSF different from the anti-M-CSF antibody ofstep (a); (d) adding an antibody to digoxigenin conjugated toperoxidase; and (e) adding a peroxidase substrate that will yield acolored reaction product that can be quantitated to determine the levelof secreted M-CSF in tissue culture cell media or a body fluid sample.Methods such as ELISA, RIA, Western blot, immunohistochemistry, andantigen capture assay are well known in the art. See, e.g., Harlow andLane, supra. In addition, the immunoassays can be scaled up for highthroughput screening in order to test a large number of compounds forinhibition or activation of M-CSF.

The anti-M-CSF antibodies of the invention can also be used to determinethe levels of cell surface M-CSF in a tissue or in cells derived fromthe tissue. In some embodiments, the tissue is from a diseased tissue.In some embodiments, the tissue can be a tumor or a biopsy thereof. Insome embodiments of the method, a tissue or a biopsy thereof can beexcised from a patient. The tissue or biopsy can then be used in animmunoassay to determine, e.g., total M-CSF levels, cell surface levelsof M-CSF, or localization of M-CSF by the methods discussed above.

The method can comprise the steps of administering a detectably labeledanti-M-CSF antibody or a composition comprising them to a patient inneed of such a diagnostic test and subjecting the patient to imaginganalysis to determine the location of the M-CSF-expressing tissues.Imaging analysis is well known in the medical art, and includes, withoutlimitation, x-ray analysis, magnetic resonance imaging (MRI) or computedtomography (CE). The antibody can be labeled with any agent suitable forin vivo imaging, for example a contrast agent, such as barium, which canbe used for x-ray analysis, or a magnetic contrast agent, such as agadolinium chelate, which can be used for MRI or CE. Other labelingagents include, without limitation, radioisotopes, such as ⁹⁹Tc. Inanother embodiment, the anti-M-CSF antibody will be unlabeled and willbe imaged by administering a second antibody or other molecule that isdetectable and that can bind the anti-M-CSF antibody. In an embodiment,a biopsy is obtained from the patient to determine whether the tissue ofinterest expresses M-CSF.

The anti-M-CSF antibodies of the invention can also be used to determinethe secreted levels of M-CSF in a body fluid such as blood serum, urine,or synovial fluid derived from a tissue. In some embodiments, the bodyfluid is from a diseased tissue. In some embodiments, the body fluid isfrom a tumor or a biopsy thereof. In some embodiments of the method,body fluid is removed from a patient. The body fluid is then used in animmunoassay to determine secreted M-CSF levels by the methods discussedabove. One embodiment of the invention is a method of assaying for theactivity of a M-CSF antagonist comprising: administering a M-CSFantagonist to a primate or human subject and measuring the number ofCD14+CD16+ monocytes in a biological sample.

Therapeutic Methods of Use

In another embodiment, the invention provides a method for inhibitingM-CSF activity by administering an anti-M-CSF antibody to a patient inneed thereof. Any of the types of antibodies described herein may beused therapeutically. In a preferred embodiment, the anti-M-CSF antibodyis a human, chimeric or humanized antibody. In another preferredembodiment, the M-CSF is human and the patient is a human patient.Alternatively, the patient may be a mammal that expresses a M-CSF thatthe anti-M-CSF antibody cross-reacts with. The antibody may beadministered to a non-human mammal expressing a M-CSF with which theantibody cross-reacts (i.e. a primate) for veterinary purposes or as ananimal model of human disease. Such animal models may be useful forevaluating the therapeutic efficacy of antibodies of this invention.

As used herein, the term “a disorder in which M-CSF activity isdetrimental” is intended to include diseases and other disorders inwhich the presence of high levels of M-CSF in a subject suffering fromthe disorder has been shown to be or is suspected of being eitherresponsible for the pathophysiology of the disorder or a factor thatcontributes to a worsening of the disorder. Such disorders may beevidenced, for example, by an increase in the levels of M-CSF secretedand/or on the cell surface or increased tyrosine autophosphorylation ofc-fms in the affected cells or tissues of a subject suffering from thedisorder. The increase in M-CSF levels may be detected, for example,using an anti-M-CSF antibody as described above.

In one embodiment, an anti-M-CSF antibody may be administered to apatient who has a c-fms-expressing tumor or a tumor that secretes M-CSFand/or that expresses M-CSF on its cell surface. Preferably, the tumorexpresses a level of c-fms or M-CSF that is higher than a normal tissue.The tumor may be a solid tumor or may be a non-solid tumor, such as alymphoma. In a more preferred embodiment, an anti-M-CSF antibody may beadministered to a patient who has a c-fms-expressing tumor, aM-CSF-expressing tumor, or a tumor that secretes M-CSF that iscancerous. Further, the tumor may be cancerous. In an even morepreferred embodiment, the tumor is a cancer of lung, breast, prostate orcolon. In another preferred embodiment, the anti-M-CSF antibodyadministered to a patient results in M-CSF no longer bound to the c-fmsreceptor. In a highly preferred embodiment, the method causes the tumornot to increase in weight or volume or to decrease in weight or volume.In another embodiment, the method causes c-fms on tumor cells to not bebound by M-CSF. In another embodiment, the method causes M-CSF on tumorcells to not be bound to c-fms. In another embodiment, the method causessecreted M-CSF of the tumor cells to not be bound to c-fms. In apreferred embodiment, the antibody is selected from 252, 88, 100, 3.8.3,2.7.3, 1.120.1, 9.14.4I, 8.10.3F, 9.7.2IF, 9.14.4, 8.10.3, 9.7.2,9.7.2C-Ser, 9.14.4C-Ser, 8.10.3C-Ser, 8.10.3-CG2, 9.7.2-CG2, 9.7.2-CG4,9.14.4-CG2, 9.14.4-CG4, 9.14.4-Ser, 9.7.2-Ser, 8.10.3-Ser, 8.10.3-CG4,8.10.3FG1 or 9.14.4G1, or comprises a heavy chain, light chain orantigen binding region thereof.

In another preferred embodiment, an anti-M-CSF antibody may beadministered to a patient who expresses inappropriately high levels ofM-CSF. It is known in the art that high-level expression of M-CSF canlead to a variety of common cancers. In one embodiment, said methodrelates to the treatment of cancer such as brain, squamous cell,bladder, gastric, pancreatic, breast, head, neck, esophageal, prostate,colorectal, lung, renal, kidney, ovarian, gynecological or thyroidcancer. Patients that can be treated with a compounds of the inventionaccording to the methods of this invention include, for example,patients that have been diagnosed as having lung cancer, bone cancer,pancreatic cancer, skin cancer, cancer of the head and neck, cutaneousor intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer,cancer of the anal region, stomach cancer, colon cancer, breast cancer,gynecologic tumors (e.g., uterine sarcomas, carcinoma of the fallopiantubes, carcinoma of the endometrium, carcinoma of the cervix, carcinomaof the vagina or carcinoma of the vulva), Hodgkin's disease, cancer ofthe esophagus, cancer of the small intestine, cancer of the endocrinesystem (e.g., cancer of the thyroid, parathyroid or adrenal glands),sarcomas of soft tissues, cancer of the urethra, cancer of the penis,prostate cancer, chronic or acute leukemia, solid tumors (e.g.,sarcomas, carcinomas or lymphomas that are cancers of body tissues otherthan blood, bone marrow or the lymphatic system), solid tumors ofchildhood, lymphocytic lymphomas, cancer of the bladder, cancer of thekidney or ureter (e.g., renal cell carcinoma, carcinoma of the renalpelvis), or neoplasms of the central nervous system (e.g., primary CNSlymphoma, spinal axis tumors, brain stem gliomas or pituitary adenomas).In a more preferred embodiment, the anti-M-CSF antibody is administeredto a patient with breast cancer, prostate cancer, lung cancer or coloncancer. In an even more preferred embodiment, the method causes thecancer to stop proliferating abnormally, or not to increase in weight orvolume or to decrease in weight or volume.

The antibody may be administered once, but more preferably isadministered multiple times. For example, the antibody may beadministered from three times daily to once every six months or longer.The administering may be on a schedule such as three times daily, twicedaily, once daily, once every two days, once every three days, onceweekly, once every two weeks, once every month, once every two months,once every three months and once every six months. The antibody may alsobe administered continuously via a minipump. The antibody may beadministered via an oral, mucosal, buccal, intranasal, inhalable,intravenous, subcutaneous, intramuscular, parenteral, intratumor ortopical route. The antibody may be administered at the site of the tumoror inflamed body part, into the tumor or inflamed body part, or at asite distant from the site of the tumor or inflamed body part. Theantibody may be administered once, at least twice or for at least theperiod of time until the condition is treated, palliated or cured. Theantibody generally will be administered for as long as the tumor ispresent provided that the antibody causes the tumor or cancer to stopgrowing or to decrease in weight or volume or until the inflamed bodypart is healed. The antibody will generally be administered as part of apharmaceutical composition as described supra. The dosage of antibodywill generally be in the range of 0.1-100 mg/kg, more preferably 0.5-50mg/kg, more preferably 1-20 mg/kg, and even more preferably 1-10 mg/kg.The serum concentration of the antibody may be measured by any methodknown in the art.

In another aspect, the anti-M-CSF antibody may be co-administered withother therapeutic agents, such as anti-inflammatory agents,anti-coagulant agents, agents that will lower or reduce blood pressure,anti-neoplastic drugs or molecules, to a patient who has ahyperproliferative disorder, such as cancer or a tumor. In one aspect,the invention relates to a method for the treatment of thehyperproliferative disorder in a mammal comprising administering to saidmammal a therapeutically effective amount of a compound of the inventionin combination with an anti-tumor agent selected from the groupconsisting of, but not limited to, mitotic inhibitors, alkylatingagents, anti-metabolites, intercalating agents, growth factorinhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors,biological response modifiers, anti-hormones, kinase inhibitors, matrixmetalloprotease inhibitors, genetic therapeutics and anti-androgens. Ina more preferred embodiment, the antibody may be administered with anantineoplastic agent, such as adriamycin or taxol. In another preferredembodiment, the antibody or combination therapy is administered alongwith radiotherapy, chemotherapy, photodynamic therapy, surgery or otherimmunotherapy. In yet another preferred embodiment, the antibody will beadministered with another antibody. For example, the anti-M-CSF antibodymay be administered with an antibody or other agent that is known toinhibit tumor or cancer cell proliferation, e.g., an antibody or agentthat inhibits erbB2 receptor, EGF-R, CD20 or VEGF.

Co-administration of the antibody with an additional therapeutic agent(combination therapy) encompasses administering a pharmaceuticalcomposition comprising the anti-M-CSF antibody and the additionaltherapeutic agent and administering two or more separate pharmaceuticalcompositions, one comprising the anti-M-CSF antibody and the other(s)comprising the additional therapeutic agent(s). Further, althoughco-administration or combination therapy generally means that theantibody and additional therapeutic agents are administered at the sametime as one another, it also encompasses instances in which the antibodyand additional therapeutic agents are administered at different times.For instance, the antibody may be administered once every three days,while the additional therapeutic agent is administered once daily.Alternatively, the antibody may be administered prior to or subsequentto treatment of the disorder with the additional therapeutic agent.Similarly, administration of the anti-M-CSF antibody may be administeredprior to or subsequent to other therapy, such as radiotherapy,chemotherapy, photodynamic therapy, surgery or other immunotherapy

The antibody and one or more additional therapeutic agents (thecombination therapy) may be administered once, twice or at least theperiod of time until the condition is treated, palliated or cured.Preferably, the combination therapy is administered multiple times. Thecombination therapy may be administered from three times daily to onceevery six months. The administering may be on a schedule such as threetimes daily, twice daily, once daily, once every two days, once everythree days, once weekly, once every two weeks, once every month, onceevery two months, once every three months and once every six months, ormay be administered continuously via a minipump. The combination therapymay be administered via an oral, mucosal, buccal, intranasal, inhalable,intravenous, subcutaneous, intramuscular, parenteral, intratumor ortopical route. The combination therapy may be administered at a sitedistant from the site of the tumor. The combination therapy generallywill be administered for as long as the tumor is present provided thatthe antibody causes the tumor or cancer to stop growing or to decreasein weight or volume.

In a still further embodiment, the anti-M-CSF antibody is labeled with aradiolabel, an immunotoxin or a toxin, or is a fusion protein comprisinga toxic peptide. The anti-M-CSF antibody or anti-M-CSF antibody fusionprotein directs the radiolabel, immunotoxin, toxin or toxic peptide tothe M-CSF-expressing cell. In a preferred embodiment, the radiolabel,immunotoxin, toxin or toxic peptide is internalized after the anti-M-CSFantibody binds to the M-CSF on the surface of the target cell.

In another aspect, the anti-M-CSF antibody may be used to treatnoncancerous states in which high levels of M-CSF and/or M-CSF have beenassociated with the noncancerous state or disease. In one embodiment,the method comprises the step of administering an anti-M-CSF antibody toa patient who has a noncancerous pathological state caused orexacerbated by high levels of M-CSF and/or M-CSF levels or activity. Ina more preferred embodiment, the anti-M-CSF antibody slows the progressof the noncancerous pathological state. In a more preferred embodiment,the anti-M-CSF antibody stops or reverses, at least in part, thenoncancerous pathological state.

Gene Therapy

The nucleic acid molecules of the instant invention can be administeredto a patient in need thereof via gene therapy. The therapy may be eitherin vivo or ex vivo. In a preferred embodiment, nucleic acid moleculesencoding both a heavy chain and a light chain are administered to apatient. In a more preferred embodiment, the nucleic acid molecules areadministered such that they are stably integrated into chromosomes of Bcells because these cells are specialized for producing antibodies. In apreferred embodiment, precursor B cells are transfected or infected exvivo and re-transplanted into a patient in need thereof. In anotherembodiment, precursor B cells or other cells are infected in vivo usinga virus known to infect the cell type of interest. Typical vectors usedfor gene therapy include liposomes, plasmids and viral vectors.Exemplary viral vectors are retroviruses, adenoviruses andadeno-associated viruses. After infection either in vivo or ex vivo,levels of antibody expression can be monitored by taking a sample fromthe treated patient and using any immunoassay known in the art ordiscussed herein.

In a preferred embodiment, the gene therapy method comprises the stepsof administering an isolated nucleic acid molecule encoding the heavychain or an antigen-binding portion thereof of an anti-M-CSF antibodyand expressing the nucleic acid molecule. In another embodiment, thegene therapy method comprises the steps of administering an isolatednucleic acid molecule encoding the light chain or an antigen-bindingportion thereof of an anti-M-CSF antibody and expressing the nucleicacid molecule. In a more preferred method, the gene therapy methodcomprises the steps of administering of an isolated nucleic acidmolecule encoding the heavy chain or an antigen-binding portion thereofand an isolated nucleic acid molecule encoding the light chain or theantigen-binding portion thereof of an anti-M-CSF antibody of theinvention and expressing the nucleic acid molecules. The gene therapymethod may also comprise the step of administering another anti-canceragent, such as taxol or adriamycin.

In order that this invention may be better understood, the followingexamples are set forth. These examples are for purposes of illustrationonly and are not to be construed as limiting the scope of the inventionin any manner.

EXAMPLE I Generation of Cell Lines Producing Anti-M-CSF Antibody

Antibodies of the invention were prepared, selected, and assayed asfollows:

Immunization and Hybridoma Generation

Eight to ten week old XENOMOUSE® transgenic mice that make humanantibodies were immunized intraperitoneally or in their hind footpadswith human M-CSF (10 μg/dose/mouse). This dose was repeated five toseven times over a three to eight week period. Four days before fusion,the mice were given a final injection of human M-CSF in PBS. The spleenand lymph node lymphocytes from immunized mice were fused with thenon-secretory myeloma P3-X63-Ag8.653 cell line, and the fused cells weresubjected to HAT selection as previously described (Galfre and Milstein,Methods Enzymol. 73:3-46, 1981). A panel of hybridomas all secretingM-CSF specific human IgG2 and IgG4 antibodies was recovered. Antibodiesalso were generated using XENOMAX® antibody selection technology asdescribed in Babcook, J. S. et al., Proc. Natl. Acad. Sci. USA93:7843-48, 1996. Nine cell lines engineered to produce antibodies ofthe invention were selected for further study and designated 252, 88,100, 3.8.3, 2.7.3, 1.120.1, 9.14.4, 8.10.3 and 9.7.2. The hybridomaswere deposited under terms in accordance with the Budapest Treaty withthe American Type Culture Collection (ATCC), 10801 University Blvd.,Manassas, Va. 20110-2209 on Aug. 8, 2003. The hybridomas have beenassigned the following accession numbers:

Hybridoma 3.8.3 (LN 15891) PTA-5390 Hybridoma 2.7.3 (LN 15892) PTA-5391Hybridoma 1.120.1 (LN 15893) PTA-5392 Hybridoma 9.7.2 (LN 15894)PTA-5393 Hybridoma 9.14.4 (LN 15895) PTA-5394 Hybridoma 8.10.3 (LN15896) PTA-5395 Hybridoma 88-gamma (UC 25489) PTA-5396 Hybridoma88-kappa (UC 25490) PTA-5397 Hybridoma 100-gamma (UC 25491) PTA-5398Hybridoma 100-kappa (UC 25492) PTA-5399 Hybridoma 252-gamma (UC 25493)PTA-5400 Hybridoma 252-kappa (UC 25494) PTA-5401

EXAMPLE II Gene Utilization Analysis

DNA encoding the heavy and light chains of monoclonal antibodies 252,88, 100, 3.8.3, 2.7.3, 1,120.1, 9.14.4, 8.10.3 and 9.7.2 was cloned fromthe respective hybridoma cell lines and the DNA sequences weredetermined by methods known to one skilled in the art. Additionally, DNAfrom the hybridoma cell lines 9.14.4, 8.10.3 and 9.7.2 was mutated atspecific framework regions in the variable domain and/orisotype-switched to obtain, for example, 9.14.4I, 8.10.3F, and 9.7.2IF,respectively. From nucleic acid sequence and predicted amino acidsequence of the antibodies, the identity of the gene usage for eachantibody chain was determined (“VBASE”). Table 2 sets forth the geneutilization of selected antibodies in accordance with the invention:

TABLE 2 Heavy and Light Chain Gene Utilization Heavy Chain Kappa LightChain Clone SEQ ID NO: V_(H) D_(H) J_(H) SEQ ID NO: V_(K) J_(K) 252 1, 23-11 7-27 6 3, 4 O12 3 88 5, 6 3-7 6-13 4 7, 8 O12 3 100  9, 10 3-231-26 4 11, 12 L2 3 3.8.3 14 3-11 7-27 4 16 L5 3 2.7.3 18 3-33 1-26 4 20L5 4 1.120.1 22 1-18 4-23 4 24 B3 1 9.14.4I 25, 26 3-11 7-27 4b 27, 28O12 3 8.10.3F 29, 30 3-48 1-26 4b 31, 32 A27 4 9.7.2IF 33, 34 3-11 6-136b 35, 36 O12 3 9.14.4 37, 38 3-11 7-27 4b 27, 28 O12 3 8.10.3 29, 303-48 1-26 4b 43, 44 A27 4 9.7.2 45, 46 3-11 6-13 6b 47, 48 O12 38.10.3FG1 97, 98 3-48 1-26 4b 31, 32 A27 4 9.14.4G1 101, 102 3-11 7-274b 27, 28 O12 3 9.14.4C-Ser 54 3-11 7-27 4b 56 O12 3 9.14.4-CG2 74 3-117-27 4b 56 O12 3 9.14.4-CG4 78 3-11 7-27 4b 56 O12 3 8.10.3C-Ser 58 3-481-26 4b 60 A27 4 8.10.3-CG2 62 3-48 1-26 4b 60 A27 4 8.10.3-CG4 94 3-481-26 4b 60 A27 4 8.10.3-Ser 90 3-48 1-26 4b 43, 44 A27 4 9.7.2C-Ser 503-11 6-13 6b 52 O12 3 9.7.2-CG2 66 3-11 6-13 6b 52 O12 3 9.7.2-CG4 703-11 6-13 6b 52 O12 3 9.7.2-Ser 86 3-11 6-13 6b 47, 48 O12 3 9.14.4-Ser82 3-11 7-27 4b 27, 28 O12 3

Mutagenesis of specific residues of the heavy and light chains wascarried out by designing primers and using the QuickChange Site DirectedMutagenesis Kit from Stratagene, according to the manufacturer'sinstructions. Mutations were confirmed by automated sequencing, andmutagenized inserts were subcloned into expression vectors. Theexpression vectors were transfected into HEK293 cells to produce enoughof the antibodies for characterization.

EXAMPLE III M-CSF Mouse Monocytic Cell Proliferation Assay

In vitro assays were conducted to measure M-CSF-dependent mousemonocytic cell proliferation in the presence of anti-M-CSF antibodies todetermine the degree of inhibition by anti-M-CSF antibodies.

Mouse monocytic cells, M-NFS-60 cells, from American Type CultureCollection (ATCC) (Manassas, Va.), were obtained and maintained inRPMI-1640 medium containing 2 mM L-glutamine (ATCC), 10% heatinactivated fetal bovine serum (FBS) (Invitrogen, Carlsbad, Calif.),0.05 mM 2-mercaptoethanol (Sigma, St. Louis Mo.) (assay medium), with 15ng/ml human M-CSF. M-NSF-60 cells were split to 5×10⁴ for next day useor to 2.5×10⁴ for use in 2 days. Prior to use in the assay, the cellswere washed three times with RPMI-1640, counted and the volume adjustedwith assay medium to yield 2×10⁵ cells/ml. All conditions were conductedin triplicate in 96-well treated tissue culture plates (Corning,Corning, N.Y.). To each well 50 μl of the washed cells, either 100 pM or1000 pM M-CSF in a volume of 25 μl and test or control antibody atvarious concentrations in a volume of 25 μl in acetate buffer (140 mMsodium chloride, 20 mM sodium acetate, and 0.2 mg/ml polysorbate 80, pH5.5) to a final volume of 100 μl was added. Antibodies of the inventionwere tested alone and with human M-CFS. The plates were incubated for 24hours (hrs) at 37° C. with 5% CO₂.

After 24 hrs, 10 μl/well of 0.5 μCi ³H-thymidine (Amersham Biosciences,Piscataway, N.J.) was added and pulsed with the cells for 3 hrs. Todetect the amount of incorporated thymidine, the cells were harvestedonto pre-wet unifilter GF/C filterplates (Packard, Meriden, Conn.) andwashed 10 times with water. The plates were allowed to dry overnight.Bottom seals were added to the filterplates. Next, 45 μl Microscint 20(Packard, Meriden, Conn.) per well was added. After a top seal wasadded, the plates were counted in a Trilux microbeta counter (Wallac,Norton, Ohio).

These experiments demonstrate that anti-M-CSF antibodies of theinvention inhibit mouse monocytic cell proliferation in response toM-CSF. Further, by using various concentrations of antibodies, the IC₅₀for inhibition of mouse nonocytic cell proliferation was determined forantibodies 252, 88, 100, 3.8.3, 2.7.3, 1.120.1, 9.14.4I, 8.10.3F,9.7.2IF, 9.14.4, 8.10.3, and 9.7.2 (Cell Proliferation Assay, Table 3aand Table 3b).

TABLE 3a Antibody 252 88 100 3.8.3 2.7.3 1.120.1 M-CSF Mouse Monocytic1.86 × 10⁻¹⁰ 2.31 × 10⁻¹⁰  7.44 × 10⁻¹⁰ 7.3 × 10⁻¹¹ 1.96 × 10⁻¹⁰ 1.99 ×10⁻¹⁰ Cell Proliferation Assay [IC₅₀, M] Human Whole Blood 8.67 × 10⁻¹⁰5.80 × 10⁻¹⁰  1.53 × 10⁻¹⁰ 8.6 × 10⁻¹¹ 7.15 × 10⁻¹⁰ 8.85 × 10⁻¹⁰Monocyte Activation Assay [IC₅₀, M] Receptor Binding 7.47 × 10⁻¹⁰ 4.45 ×10⁻¹⁰ 1.252 × 10⁻⁹ 7.0 × 10⁻¹¹ 3.08 × 10⁻¹⁰ 1.57 × 10⁻¹⁰ InhibitionAssay [IC₅₀, M]

TABLE 3b Antibody 9.14.4I 8.10.3F 9.7.2IF 9.14.4 8.10.3 9.7.2 M-CSFMouse 2.02 × 10⁻¹⁰ 4.13 × 10⁻¹⁰  7.37 × 10⁻¹⁰ 2.02 × 10⁻¹⁰ 4.13 × 10⁻¹⁰7.37 × 10⁻¹⁰ Monocytic Cell Proliferation Assay [IC₅₀, M] Human WholeBlood 2.49 × 10⁻¹⁰ 4.46 × 10⁻¹⁰ 1.125 × 10⁻⁹ 6.48 × 10⁻¹⁰  2.8 × 10⁻¹⁰1.98 × 10⁻¹⁰ Monocyte Activation Assay [IC₅₀, M] Receptor Binding 2.97 ×10⁻¹⁰  9.8 × 10⁻¹¹  5.29 × 10⁻¹⁰  4.1 × 10⁻¹¹  1.5 × 10⁻⁹   6 × 10⁻¹²Inhibition Assay [IC₅₀, M ]

EXAMPLE IV Human Whole Blood Monocyte Activation Assay

In vitro assays were conducted to measure M-CSF dependent monocyte shapechanges in the presence of anti-M-CSF antibodies to determine if theanti-M-CSF antibodies were capable of inhibiting whole blood monocyteactivation and their degree of inhibition of monocyte shape changes.

In individual wells of a 96-well tissue culture plate, 6 μl of 1.7 nManti-M-CSF and 94 μl of whole human blood for a final concentration of102 pM anti-M-CSF antibody were mixed. The plates were incubated at 37°C. in a CO₂ tissue culture incubator. Next, the plates were removed fromthe incubator. To each well, 100 μl of a fixative solution (0.5%formalin in phosphate buffered saline without MgCl₂ or CaCl₂) was addedand the plates were incubated for 10 minutes at room temperature. Foreach sample, 180 μl from each well and 1 ml of Red Cell Lysis Bufferwere mixed. The tubes were vortexed for 2 seconds. Next, the sampleswere incubated at 37° C. for 5 minutes in a shaking water bath to lysethe red blood cells, but to leave monocytes intact. Immediatelyfollowing this incubation, the samples were read on afluorescence-activated cell scanning (FACS) machine (BD Beckman FACS)and data was analyzed using FACS Station Software Version 3.4.

These experiments demonstrate that anti-M-CSF antibodies of theinvention inhibit monocyte shape changes compared to control samples.Using the monocyte shape change assay, the IC₅₀ was determined forantibodies 252, 88, 100, 3.8.3, 2.7.3, 1.120.1, 9.14.4I, 8.10.3F,9.7.2IF, 9.14.4, 8.10.3, and 9.7.2 (Human Whole Blood MonocyteActivation, Table 3a and Table 3b).

EXAMPLE V c-Fms Receptor Binding Inhibition Assay

In vitro assays were conducted to measure M-CSF binding to c-fmsreceptor in the presence of anti-M-CSF antibodies to determine if theanti-M-CSF antibodies were capable of inhibiting M-CSF binding to c-fmsreceptor and their degree of inhibition.

NIH-3T3 cells transfected with human c-fms or M-NSF-60 cells maintainedin Dulbecco's phosphate buffered saline without magnesium or calciumwere washed. NIH-3T3 cells were removed from tissue culture plates with5 mM ethylene-diamine-tetra-acetate (EDTA), pH 7.4. The NIH-3T3 cellswere returned to the tissue culture incubator for 1-2 minutes and theflask(s) were tapped to loosen the cells. The NIH-3T3 cells and theM-NSF-60 cells were transferred to 50 ml tubes and washed twice withreaction buffer (lx RPMI without sodium bicarbonate containing 50 mMN-2-Hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.4).Next, the NIH-3T3 cells were resuspended in reaction buffer for a finalconcentration of 1.5×10⁵ cell/ml. The M-NSF-60 cells were resuspended ina reaction buffer for a final concentration of 2.5×10⁶ cells/ml.

For the assay, 9 μl of a sterile 0.4 M sucrose solution, 100 μl of¹²⁵I-M-CSF (Amersham, IMQ7228v) at a final concentration of 200 pM inRPMI-1640 containing 50 mM HEPES (pH 7.4), 0.2% bovine serum albumin,and 100 μl of unlabeled M-CSF at a final concentration of 200 nM weremixed in a binding tube. Next, 50 μl/tube of increasing concentrationsof a test antibody was added. In order to determine non-specific bindingof the antibodies, we included samples to which we also added 200 nMM-CSF. To control tubes, we did not add antibody. Next, 15,000 NIH-3T3cells or 250,000 M-NSF-60 cells were added per tube. All tubes wereincubated at room temperature for 3 hrs and subjected to centrifugationat 10,000 rpm for 2 min. The tips of the tubes containing the cellpellets were cut off and the amount of M-CSF bound to the cells wasdetermined using a Packard Cobra II Gamma counter. The specific bindingwas determined by subtracting non-specific binding from total binding.All assays were performed in duplicate. The binding data was analyzedusing the computer program, Graph Pad Prism 2.01.

These experiments demonstrate that anti-M-CSF antibodies of theinvention inhibit the binding of M-CSF to c-fms receptor compared tocontrol samples. Further, by using various concentrations of antibodies,the IC₅₀ for inhibition of receptor binding was determined forantibodies 252, 88, 100, 3.8.3, 2.7.3, 1.120.1, 9.14.4I, 8.10.3F,9.7.2IF, 9.14.4, 8.10.3, and 9.7.2 (Receptor Binding Inhibition Assay,Table 3a and Table 3b).

EXAMPLE VI Determination of Affinity Constants (K_(D)) of Anti-M-CSFMonoclonal Antibodies by BIACORE™

Affinity measures of purified antibodies were performed by surfaceplasmon resonance using the BIACORE™ 3000 instrument, following themanufacturer's protocols.

For antibodies 3.8.3, 2.7.3 and 1.120.1, the experiments were performedin a BIACORE™ 3000 instrument at 25° C. in Dulbecco's phosphate bufferedsaline containing 0.0005% Tween-20. Protein concentrations were obtainedfrom sedimentation velocity experiments or by measuring the wavelengthof the sample at 280 nm using theoretical extinction coefficientsderived from amino acid sequences. For experiments measuring the bindingof antibody to immobilized antigens, M-CSF was immobilized on a B1 chipby standard direct amine coupling procedures. Antibody samples wereprepared at 0.69 μM for 3.8.3, 2.7.3 and 1.120.1. These samples werediluted 3-fold serially to 8.5 nM or 2.8 nM for roughly a 100-fold rangein concentrations. For each concentration, the samples were injected induplicate at 5 μl/min flow for 4 min. The dissociation was monitored for2000 seconds. The data were fit globally to a simple 1:1 binding modelusing BIACORE™ Biaevaluation software. In all cases, this method wasused to obtain k_(off) and it was found that this data set compared wellto data obtained from global fit of association and dissociation data.

For antibodies 252, 88 and 100, the experiments were performed in aBIACORE™ 3000 instrument at 25° C. in HBS-EP Buffer (0.01M HEPES, pH7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20). For experimentsmeasuring the binding of antibody to immobilized antigens, a M-CSF wasimmobilized on a CM5 Research Grade Sensor chip by standard direct aminecoupling procedures. Antibody samples were prepared at 12.5 nM forantibodies 252 and 100 and at 25.0 nM for antibody 88. These sampleswere two-fold serially diluted to 0.78 nM for roughly a 15-30 fold rangein concentrations. For each concentration, the samples were injected induplicate in random order at 30 μl/min flow for 3 min. The dissociationwas monitored for 300 sec. The data were fit globally to a simple 1:1binding model using BIACORE™ Biaevaluation software. In all cases, thismethod was used to obtain k_(off) and it was found that this data setcompared well to data obtained from global fit of association anddissociation data.

Table 4 shows results for antibodies 252, 88, 100, 3.8.3, 2.7.3 and1.120.1.

TABLE 4 252 88 100 3.8.3 2.7.3 1.120.1 K_(D) (M) 1.33 × 1.33 × 2.0 × 4.0× 4.7 × 5.4 × 10⁻¹¹ 10⁻⁹ 10⁻¹¹ 10⁻¹⁰ 10⁻¹⁹ 10⁻⁹ k_(off) (1/s) 1.03 × 7.3 × 1.7 × 10⁻⁶ 10⁻⁵ 10⁻⁵

EXAMPLE VII Production of 8.10.3 Antibodies from 8.10.3 Hybridoma Cells

Antibody 8.10.3 was produced in 3 L sparged spinners. The 3 L spargedspinner flask is a glass vessel where cultures are mixed with animpeller controlled by a magnetic platform. The spinner is connected togas lines to provide 5% CO₂ and air. 8.10.3 hybridoma cells wereinitially thawed into T-25 cell culture flasks. The cells wereprogressively expanded until there was a sufficient number of cells toseed the sparged spinners.

Two 3 L sparged spinner flasks were seeded with 8.10.3 hybridoma cellsin Hybridoma Serum-Free Medium with the additions noted on Table 5, forthe two sparged flasks. The concentrations for Ultra low IgG serum(Gibco cat#16250-078), L-glutamine (JRH Biosciences cat#59202-500M),Non-Essential Amino Acids (Gibco cat#11140-050), Peptone (Difcocat#211693), glucose (In-house stock prepared from JT Bakercat#1920-07), and Anti-foam C (Sigma cat.# A-8011) are given at theirfinal concentrations in the media. The balance of the volume in eachreactor is Hybridoma Serum-Free Medium.

TABLE 5 Conditions for Growing Hybridoma 8.10.3 in two 3L spargedspinners. Conditions Spinner 1 Spinner 2 Seeding density (1 × 10⁶cells/ml) 0.16 ml 0.16 ml Hybridoma Serum-Free Medium Balance Balance(Gibco cat# 12045-076) Ultra low IgG serum 5% 5% (Gibco cat# 16250-078)L-glutamine (JRH Biosciences 8 mmol/L 8 mmol/L cat# 59202-500M)Non-Essential Amino Acids 1% 1% (Gibco cat# 11140-050) Peptone (Difcocat# 211693) 1 g/L 1 g/L 2M glucose (In-house stock prepared 8 g/L 8 g/Lfrom JT Baker cat# 1920-07) Anti-foam C (Sigma cat.# A-8011) 1 ml/L 1ml/L

The cultures were grown for 15 days and were harvested when theviability was below 20%. Viability was determined by trypan blueexclusion method with an automated cell counter (Cedex, Innovatis).Harvesting was accomplished by centrifugation and subsequent filtration.Clarified supernatant was obtained after centrifugation for 15 minutesat 7000 rpm and subsequent filtration with a sterile 0.22 μm 4″ OpticapMillipore filter (cat# KVSCO4HB3) into a 10 L sterile TC-Tech bag (cat #P/N 12420 Bag Style CC-10-112420). The filtrate was then purified in thefollowing example.

EXAMPLE VIII Purification of an Anti-M-CSF Antibody

A Protein A column (Amersham Pharmacia) was prepped by washing with 3column volumes of 8M Urea, followed by an equilibration wash with 20 mMTris (pH 8). The final filtrate from Example VII was spiked with 2% v/vof 1M Tris pH 8.3 and 0.02% NaN₃ before being loaded onto the Protein Acolumn via gravity-drip mode. After load was complete, the resin waswashed with 5 column volumes of 20 mM Tris (pH 8), followed by 5 columnvolumes of the elution buffer (0.1 M Glycine pH 3.0). Any precipitationwas noted, and then a 10% v/v spike of 1M Tris pH 8.3 was added to theeluted antibody. The eluted protein was then dialyzed into 100 fold thevolume amount of eluted material of dialysis buffer (140 mM NaCl/20 mMSodium Acetate pH 5.5). Following dialysis, the antibody was sterilefiltered with a 0.22 μm filter and stored until further use.

EXAMPLE IX Monkey Treatment and Monocyte Counts

One male and one female cynomolgus monkey per dosage group wereintravenously administered vehicle or antibody 8.10.3 (produced asdescribe in Examples VII and VIII) at 0, 0.1, 1, or 5 mg/kg in a dosevolume of 3.79 mL/kg over an approximately 5 minute period. Bloodsamples for clinical laboratory analysis were collected at 24 and 72hours postdose and weekly for 3 weeks. The monocyte counts weredetermined by light scatter using an Abbott Diagnostics Inc. Cell Dynsystem (Abbott Park, Ill.).

A dose-related decrease (˜25% to 85%) in total monocytes at all doses(FIGS. 1A and 1B) was observed. Monocyte counts at the 0.1 and 1 mg/kgappeared to rebound to near control levels by week 2, while monocytecounts at 5 mg/kg were still decreased at 3 weeks.

CD14+CD16+ Monocyte Subset Analysis

Primate whole blood was drawn into Vacutainer tubes containing sodiumheparin. 0.2 ml of each blood sample was added to a 15 ml conicalpolypropylene centrifuge tube containing 10 ml of red blood cell lysisbuffer (Sigma), and incubated in a 37° C. water bath for 15 minutes. Thetubes were then centrifuged in a Sorvall RT7 centrifuge for 5 minutes at1,200 rpm. The supernatant was aspirated, the pellet resuspended in 10ml of 4° C. FACS buffer (Hanks' Balanced Salt Solution/2% FBS/0.02%sodium azide), and the tube centrifuged again for 5 minutes at 1,200rpm. The supernatant was aspirated and the pellet resuspended in anantibody cocktail consisting of 80 μl 4° C. FACS buffer, 10 μlFITC-conjugated anti-human CD14 monoclonal antibody (BD Biosciences, SanDiego, Calif.), 0.5 μl Cy5-PE-conjugated anti-human CD16 monoclonalantibody (BD Biosciences, San Diego, Calif.), and 10 μl PE-conjugatedanti-human CD89 monoclonal antibody (BD Biosciences, San Diego, Calif.).The cell suspension was incubated on ice for 20 minutes, after which 10ml of 4° C. FACS buffer was added and the cells centrifuged as before.The supernatant was aspirated, and the cell pellet resuspended in 400 μlFACS buffer and the cells analyzed on a FACSCaliber flow cytometer (BDBiosciences, San Jose, Calif.). Data for 30,000 cells were collectedfrom each sample.

The monocyte population was identified by a combination of forward anglelight scatter and orthogonal light scatter. Cells within the monocytegate were further analyzed for expression of CD14 and CD16. Two distinctpopulation of monocytes were observed, one expressing high levels ofCD14 with little or no CD16 expression (CD14++CD16−) and the otherexpressing lower levels of CD14, but high levels of CD16 (CD14+CD16+),similar to the two monocyte subsets previously described in humanperipheral blood (Ziegler-Heitbrock H. W., Immunology Today 17:424-428(1996)). For each primate tested, the percentage of monocytes within theCD14+CD16+ subset was determined after each blood draw, on days 1, 3, 7,14, and 21 after 8.10.3 injection.

In general, 8.10.3 treatment resulted in a reduction in the percentageof CD14+CD16+ monocytes (see FIGS. 2A and 2B). Monkeys not receiving8.10.3 Antibody demonstrated relatively stable CD14+CD16+ monocytelevels. CD14+CD16+ monocytes have been termed “proinflammatory” becausethey produce higher levels of TNF-α and other inflammatory cytokines(Frankenberger, M. T., et al., Blood 87:373-377 (1996)). It has alsobeen reported that the differentiation of monocytes from theconventional CD14++CD16− phenotype to the proinflammatory phenotype isdependent on M-CSF (Saleh M. N., et al., Blood 85: 2910-2917 (1995)).

EXAMPLE X Monkey Treatment and Monocyte Counts

Three male cynomolgus monkeys per dosage group were intravenouslyadministered vehicle (20 mM Sodium acetate, pH 5.5, 140 mM NaCl),purified antibody 8.10.3F, or purified antibody 9.14.4I at 0, 1, or 5mg/kg in a dose volume of 3.79 mL/kg over an approximately 5 minuteperiod. The monkeys were 4 to 9 years of age and weighed 6 to 10 kg.Blood samples for clinical laboratory analysis were collected at 2, 4,8, 15, 23, and 29 days. Monocyte counts were determined by light scatterusing an Abbott Diagnostics Inc. Cell Dyn system (Abbott Park, Ill.).

A decrease in the percentage change in total monocytes at all doses ofantibody 8.10.3F as compared to pre-test levels of monocytes (FIG. 3)was observed (see e.g., day 4, 8, 15, and 23 in FIG. 3).

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims.

SEQUENCES  SEQ ID NO: 1  252 Heavy Chain [Gamma chain]nucleotide sequence atggagttggggctgtgctggattaccttgttgctattataaaaggtgtccagtgtCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATTTCATACATTAGTGGTAGTGGTAGTACCATATACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATCACTGTGCGAGAGCCCTGGGTGGGATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTtccaccaagggcccatccgtcttccccctggcgccctgctctagaagcacctccgagagcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatgagtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatga tctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtac gtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtca gcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctccca gcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatccc gggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtgga gtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcc tctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggct ctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa  SEQ ID NO: 2 252 Heavy Chain [Gamma chain] protein sequence melglcwiflvaiikgvqcQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIR QAPGKGLEWISYISGSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYHCARALGGMDVWGQGTTVTVSSAstkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssnfgtqtytcnvdhkpsntkvdktverkccvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsvltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkSEQ ID NO: 3  252 Light Chain [Kappa chain] nucleotide sequence atgagggtccctgctcagctcctggggctcctgctactctggctccgaggtgccagatgtGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCGGCTTTTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTACATCCAGTTTGCAAAGTGGGGTCCCATTCAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGAGTTACAGTGTCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGAactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgctagcgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctc agcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcc tgagctcgcccgtcacaaagagcttcaacaggggagagtgt  SEQ ID NO: 4 252 Light Chain [Kappa chain] protein sequence mrvpaqllgllllwlrgarcDIQMTQSPSSLSASVGDRVTITCRASQSISGFLNWYQQKPGKAPKLLIYATSSLQSGVPFRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSVPFTFGPGTKVDIKRtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgec SEQ ID NO: 5 88 Heavy Chain [Gamma chain] nucleotide sequence atggaatttgggctgtgctgggttttccttgttgctattttagaaggtgtccagtgtGAGGTGCAGCTGGTG GAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCC TGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGGATGAGCTGGGTCC GCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAGCAA GATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACC ATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGC CTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCTCCGGGTATAGCA GCAGCTGGTAGGGCCTACTGGGGCCAGGGAACCCTGGTCACCGTCTCC TCAGCTtccaccaagggcccatccgtcttccccctggcgccctgctctagaagcacctccgagagcacagcggc cctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcg tgcacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttc ggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaat gttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaagg acaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtcca gttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacg ttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaac aaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacacc ctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcg acatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccg acggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccg tgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa SEQ ID NO: 6  88 Heavy Chain [Gamma chain] protein sequence mefglcwvflvailegvqcEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWV RQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSL  RAEDTAVYYCAPGIAAAGRAY VVGQGTLVTVSSAstkgpsvfplapcsrstsestaalgcl vkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtypssnfgtqtytcnvdhkpsntkvdktverkccv ecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfns tfrvvsvltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfyp sdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk SEQ ID NO: 7  88 Light Chain [Kappa chain] nucleotide sequence atgagggtccctgctcagctcctggggctcctgctactctggctccgaggtgccagatgtGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTTGGAGACAGAGTCACCATCACTTGCCGGCCAAGTCAGGACATTAGCAGTTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATTAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGAactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttganatctggaactgctagcgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggt ggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctc agcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcc tgagctcgcccgtcacaaagagcttcaacaggggagagtgt  SEQ ID NO: 8 88 Light Chain [Kappa chain] protein sequence mrvpaqllgllllwlrgarcDIQMTQSPSSLSASVGDRVTITCRPSQDISSYLNWYQQKPGKAPKLLIYAASSLQSGVPLRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTFGPGTKVDIKRtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgec SEQ ID NO: 9 100 Heavy Chain [Gamma chain] nucleotide sequence atggagtttgggctccgctggatttttcttgtggctattttaaaaggtgtccagtgtGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGGTCTCAGCTATTAGTGGTCGTGGTGGTAGGACATACTTCGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTTCTGTGCGGTAGAAGGCTATAGTGGGCGCTACGGATTTTTTGACTACTGGGGCCAGGGAACCCTAGTCACCGTCTCCTCAGCCtccaccaagggcccatcggtcttccccctggcgccctgctctagaagcacctccgagagcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgcc ctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagaca gttgagcgcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccc caaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggiggtggacgtgagccacgaaga ccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagca gttcaacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtg caaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaacc acaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggc ttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctccca tgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtc ttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa SEQ ID NO: 10  100 Heavy Chain [Gamma chain] protein sequence mefglrwiflvailkgvqcEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGRGGRTYFADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYFCAVEGYSGRYGFFDYWGQGTLVTVSSAstkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssnfgtqtytcnvdhkpsntkvdktverkccvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsyltvvhqdwingkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqks1s1spgk SEQ ID NO: 11  100 Light Chain [Kappa chain] nucleotide sequence atggaagccccagctcagcnctcttcctcctgctactctggctcccagataccactggaGAAATAGTGATG ACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACC CTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACC AGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAC CAGGGCCAGTGGTATCCCAGACAGGATCAGTGGCAGTGGGTCTGGAAC AGAGTTCACTCTCATCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTT TATTACTGTCAGCAGTCTAATAACTGGCCATTCACTTTCGGCCCTGGGA CCAAAGTGGATATCAAACGAactgtggctgcaccatctgtcttcatcttcccgccatctgatgagca gttgaaatctggaactgctagcgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtg gataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctca gcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcct gagctcgcccgtcacaaagagcttcaacaggggagagtgt  SEQ ID NO: 12 100 Light Chain [Kappa chain] protein sequence meapaqllfllllwlpdttgEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRASGIPDRISGSGSGTEFTLIISSLQSEDFAVYYCQQSNNWPFTFGPGTKVDIKRtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgec SEQ ID NO: 14 3.8.3 Heavy Chain [Gamma chain] protein sequence mefglswvflvaiikgvqcQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWFSYISSSGSTIYYADSVKGRFTISRDNAKNSLSLQMNSLRA EDTAVYYCARGLTGDYWGQGTLVTVSSAstkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssnfgtqtytcnvdhkpsntkvdktverkccvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsyltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavew esngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk SEQ ID NO: 16  3.8.3 Light Chain [Kappa chain] protein sequence mdmrvpaqllgllllwfpgsrcDIQMTQSPSSVSASVGDRVTISCRASQDISGWLAWYQQKPGKAPKLLISATSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTNSFPFTFGPGTKVDIKRtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgecSEQ ID NO: 18  2.7.3 Heavy Chain [Gamma chain] protein sequence mefglswvflvallrgcqcQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWV RQAPGKGLEWVAFIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYRVYFDYWGQGTLVTVSSAstkgpsvfplapcsrstsestaalgclvkdyfpepvtyswnsgaltsgyhtfpaylqssglyslssyytypssslgtktytcnvdhkpsntkvdkrveskygppcpscpapeflggpsvflfppkpkdtlmisrtpevtcvvvdvsqedpevqfnwyvdgvevhnaktkpreeqfnstyrvvsyltvlhqdwingkeykckvsnkglpssiektiskakgqprepqvytlppsqeemtknqvsltclykgfypsdiavewesngqpennykttppvldsdgsfflysrltvdksrwqegnvfscsvmhealhnhytqkslslspgkSEQ ID NO: 20 2.7.3 Light Chain [Kappa chain] protein sequence mdmrypaqllgllllwfpgsrcDIQMTQSPSSVSASVGDRVTITCRASQDISSWLAWYQRKPGKAPKLQIYAASSLESGVPSRFNGSGSGTDFTLSISSLQPEDFATYYCQQTNSFPLTFGGGTKVEIKRtvaapsyfifppsdeqlksgtasATvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyaceythqglsspytksfnrgecSEQ ID NO: 22  1.120.1 Heavy Chain [Gamma chain] protein sequence mewtwsflflvaaatgahsQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQD RVTMTTDTSTTTAYMELRS LRSDDTAVYYCARRAYGANFFDYWGQGTLVTVSSAstkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtypssnfgtqtytcnvdhkpsntkvdktverkccvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyydgvevhnaktkpreeqfnstfrvvsyltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk  SEQ ID NO: 24  1.120.1 Light Chain [Kappa chain] protein sequence mylqtqvfislllwisgaygDIVMTQSPDSLAVSLGERATINCKSSQSILFFSNNKNYLAWYRQKPGQPPNLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSSPWTFGQGTKVEIKRtvaapsvfifppsdeqlksgtasvvclnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgecSEQ ID NO: 25  9.14.41 Heavy Chain [Gamma Chain] nucleotide sequence atggagtttgggctgagctgggttttccttgttgctattataaaaggtgtCCAGTGTCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTATATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGGTTTCATACATTAGTAGTAGTGGTAGTACCATATACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGGCCTAACTGGGGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTtccaccaagggcccatccgtcttccccctggcgccctgctctagaagcacctccgagagcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatgttgtgtcgag tgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatg atctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggta cgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtc agcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctccc agcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcc cgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtgg agtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttc ctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggc tctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa  SEQ ID NO: 26 9.14.41 Heavy Chain [Gamma Chain] protein sequence mefglswvflvaiikgvqcQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCARGLTGDYWGQGTLVTVSSAstkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssnfgtqtytcnvdhkpsntkvdktverkccvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsvltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkSEQ ID NO: 27 9.14.4, 9.14.4I, 9.14.4-Ser and 9.14.4-G1 Light Chain [Kappa Chain]nucleotide  sequence atggacatgagggtccccgctcagctcctggggctcctgctactctggctccgaggtgccagatgTGACATCC AGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTCGGAGACAGAGT CACCATCACTTGCCGGCCAAGTCAGATCATTAGCAGTTTATTAAATTGG TATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCCATGCTGCA TCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTG GGACAGATTTCACTCTCACCATCAGTAGTCTGCAACCTGAAGATTTTGC AACTTACTACTGTCAACAGAGTTACAGTACCCCATTCACTTTCGGCCCT GGGACCAAAGTGGATATCAAACGAactgtggctgcaccatctgtcttcatcttcccgccatctga tgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtgga aggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctaca gcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatca gggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt  SEQ ID NO: 28 9.14.4, 9.14.4I, 9.14.4-Ser and 9.14.4-G1 Light Chain [Kappa Chain]protein  sequence mdmrvpaqllgllllwlrgarcDIQMTQSPSSLSASVGDRVTITCRPSQIISSLLNWYQ QKPGKAPKLLIHAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTFGPGTKVDIKRtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgecSEQ ID NO: 37  9.14.4 Heavy Chain [Gamma Chain] nucleotide sequence atggagtttgggctgagctgggttttccttgttgctattataaaaggtgtCCAGTGTCAGGTGCAGCTG GTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTC TCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTATATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGGTTTCATACATTAGTAGTAGTGGTAGTACCATATACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGGCCTAACTGGGGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTtccaccaagggcccatccgtcttccccctggcgccctgctctagaagcacctccgagagcacagcggccctgggctgcct ggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcc cagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacgaaga cctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagagagttgagtccaaatatggtccccca tgcccatcatgcccagcacctgagttcctggggggaccatcagtcttcctgttccccccaaaacccaaggacactctca tgatctcccggacccctgaggtcacgtgcgtggtggiggacgtgagccaggaagaccccgaggtccagttcaactgg tacgtggatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtgg tcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctc ccgtcctccatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccat cccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcnctaccccagcgacatcgccgt ggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttc ttcctctacagcaggctaaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgag gctctgcacaaccactacacacagaagagcctctccctgtctccgggtaaa  SEQ ID NO: 38 9.14.4 Heavy Chain [Gamma Chain] protein sequence mefglswvflvaiikgvqcQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCARGLTGDYWGQGTLVTVSSAstkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtypssslgtktytcnvdhkpsntkvdkrveskygppcpscpapeflggpsvflfppkpkdtlmisrtpevtcvvvdvsqedpevqfnwyvdgvevhnaktkpreeqfnstyrvvsvltvlhqdwlngkeykckvsnkglpssiektiskakgqprepqvytlppsqeemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflysrltvdksrwqegnvfscsvmhealhnhytqkslslspgkSEQ ID NO: 54  9.14.4C-Ser Heavy Chain [Gamma chain] protein sequence mefglswvflvaiikgvqcQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCARGLTGDYWGQGTLVTVSSAstkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtktytcnvdhkpsntkvdkrveskygppcppcpapeflggpsvflfppkpkdtlmisrtpevtcvvvdvsqedpevqfnwyydgvevhnaktkpreeqfnstyrvvsvltylhqdwlngkeykckvsnkglpssiektiskakgqprepqvytlppsqeemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflysrltvdksrwqegnvfscsvmhealhnhytqkslslspgkSEQ ID NO: 56 9.14.4C-Ser, 9.14.4-CG2 and 9.14.4-CG4 Light Chain [Kappa chain]protein  sequence mdmrvpaqllgllllwlrgarcDIQMTQSPSSLSASVGDRVTITCRPSQIISSLLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTFGPGTKVDIKRtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgecSEQ ID NO: 74  9.14.4-CG2 Heavy Chain [Gamma chain] protein sequence mefglswvflvaiikgvqcQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCARGLTGDYWGQGTLVTVSSAstkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssnfgtqtytcnvdhkpsntkvdktverkccvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsvltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk SEQ ID NO: 78  9.14.4-CG4 Heavy Chain [Gamma chain] protein sequence mefglswvflvaiikgvqcQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCARGLTGDYWGQGTLVTVSSAstkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtktytcnvdhkpsntkvdkryeskygppcpscpapeflggpsvflfppkpkdtlmisrtpevtcvvvdvsqedpevqfnwyvdgvevhnaktkpreeqfnstyrvvsvltvlhqdwlngkeykckvsnkglpssiektiskakgqprepqvytlppsqeemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflysrltvdksrwqegnvfscsvmhealhnhytqkslslspgk SEQ ID NO: 82  9.14.4-Ser Heavy Chain [Gamma chain] protein sequence mefglswvflvaiikgvqcQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCARGLTGDYWGQGTLVTVSSAstkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtktytcnvdhkpsntkvdkrveskygppcppcpapeflggpsvflfppkpkdtlmisrtpevtcvvvdvsqedpevqfnwyvdgvevhnaktkpreeqfnstyrvvsvltvlhqdwlngkeykckvsnkglpssiektiskakgqprepqvytlppsqeemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflysrltvdksrwqegnvfscsvmhealhnhytqkslslspgkSEQ ID NO. 101  9.14.4G1 Heavy chain (gamma chain) nucleotide sequence atggagtttgggctgagctgggttttccttgttgctattataaaaggtgtccagtgtCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTATATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGGTTTCATACATTAGTAGTAGTGGTAGTACCATATACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGGCCTAACTGGGGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTtccaccaag ggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaa ggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctg tcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacat ctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcaca catgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacacc ctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaa ctggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccg tgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggictccaacaaag ccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcc cccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcg ccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggct ccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgc atgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatag SEQ ID NO 102  9.14.4G1 Heavy chain (gamma chain) protein sequence mefglswvflvaiikgvqcQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCARGLTGDYWGQGTLVTVSSAstkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwingkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkSEQ ID NO: 29  8.10.3 and 8.10.3F Heavy Chain [Gamma chain]nucleotide sequence atggagttggggctgtgctgggttttccttgagctattttagaaggtgtccagtgtGAGGTGCAGCTGGTG GAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCC TGTGCAGCCTCTGGATTCACCTTCAGTAGTTTTAGTATGACCTGGGTCC GCCAGGCTCCAGGAAAGGGGCTGGAGTGGGTTTCATACATTAGTAGTA GAAGTAGTACCATATCCTACGCAGACTCTGTGAAGGGCCGATTCACCA TCTCCAGAGACAATGCCAAGAACTCACTGTATCTGCAAATGAACAGCC TGAGAGACGAGGACACGGCTGTGTATTACTGTGCGAGAGATCCTCTTCT AGCGGGAGCTACCTTCTTTGACTACTGGGGCCAGGGAACCCTGGTCAC CGTCTCCTCAGCCtccaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgag agcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctct gaccagcggcgtgcacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgcc ctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagaca gttgagcgcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccc caaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggiggtggacgtgagccacgaaga ccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagca gttcaacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtg caaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaacc acaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggc ttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctccca tgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtc ttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa SEQ ID NO: 30  8.10.3 and 8.10.3F Heavy Chain [Gamma chain]protein sequence  melglcwvflvailegvqcEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFSMTWV RQAPGKGLEWVSYISSRSSTISYADSVKGRFTISRDNAKNSLYLQMNSLRD EDTAVYYCARDPLLAGATFFDYWGQGTLVTVSSAstkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssnfgtqtytcnvdhkpsntkvdktverkccvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsyltvvhqdwingkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkSEQ ID NO: 31  8.10.3FG1 and 8.10.3F Light Chain [Kappa chain]nucleotide sequence atggaaaccccagcgcagcttctcttcctcctgctactctggctcccagataccaccggaGAATTTGTGTTG ACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCC TCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGTTACTTAGCCTGGTA CCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCC AGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGG ACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAG TGTATTACTGTCAGCAGTATGGTAGCTCACCTCTCACTTTCGGCGGAGG GACCAAGGTGGAGATCAAACGAactgtggctgcaccatctgtcttcatcttcccgccatctgatga gcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaag gtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcc tcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcaggg cctgagctcgcccgtcacaaagagcttcaacaggggagagtgt  SEQ ID NO: 32 8.10.3FG1 and 8.10.3F Light Chain [Kappa chain] protein sequence metpaqllfllllwlpdttgEFVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKRtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgec SEQ ID NO: 43 8.10.3 and 8.10.3-Ser Light Chain [Kappa chain] nucleotide sequence atggaaaccccagcgcagcttctcttcctcctgctactctggctcccagataccaccggaGAATTTGTGTTG ACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCC TCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGTTACTTAGCCTGGTA CCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCC AGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGG ACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGTAG TGTATTACTGTCAGCAGTATGGTAGCTCACCTCTCACTTTCGGCGGAGG GACCAAGGTGGAGATCAAACGAactgtggctgcaccatctgtcttcatcttcccgccatctgatga gcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaag gtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcc tcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcaggg cctgagctcgcccgtcacaaagagcttcaacaggggagagtgt  SEQ ID NO: 44 8.10.3 and 8.10.3-Ser Light Chain [Kappa chain] protein sequence metpaqllfllllwlpdttgEFVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFVVYYCQQYGSSPLTFGGGTKVEIKRtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgec SEQ ID NO: 58 8.10.3C-Ser Heavy Chain [Gamma chain] protein sequence melglcwvflvailegvqcEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFSMT WVRQAPGKGLEWVSYISSRSSTISYADSVKGRFTISRDNAKNSLYLQMNSLRD EDTAVYYCARDPLLAGATFFDYWGQGTLVTVSSAstkgpsvfplapcsrstsestaalgclykdyfpepytvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtktytcnvdhkpsntkvdkrveskygppcppcpapeflggpsvflfppkpkdtlmisrtpevtcvvvdvsqedpevqfnwyvdgvevhnaktkpreeqfnstyrvvsvltvlhqdwingkeykckvsnkglpssiektiskakgqprepqvytlppsqeemtknqvsltclykgfypsdiavewesngqpennykttppvldsdgsfflysrltvdksrwqegnyfscsvmhealhnhytqkslslspgkSEQ ID NO: 60 8.10.3-CG2, 8.10.3-CG4 and 8.10.3C-Ser Light Chain [kappa chain]protein  sequence metpaqllfllllwlpdttgEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKRtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyaceythqglsspytksfnrgec SEQ ID NO: 62 8.10.3-CG2 Heavy Chain [Gamma chain] protein sequence melglcwvflvailegvqcEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFSMT WVRQAPGKGLEWVSYISSRSSTISYADSVKGRFTISRDNAKNSLYLQMNSLRD EDTAVYYCARDPLLAGATFFDYWGQGTLVTVSSAstkgpsvfplapcsrstsestaalgclykdyfpepytyswnsgaltsgyhtfpaylqssglyslssyytypssnfgtqtytcnvdhkpsntkvdktverkccvecppcpappyagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyydgvevhnaktkpreeqfnstfrvvsvltvvhqdwingkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkSEQ ID NO: 90  8.10.3-Ser Heavy Chain [Gamma chain] protein sequence melglcwvflvailegvqcEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFSMT WVRQAPGKGLEWVSYISSRSSTISYADSVKGRFTISRDNAKNSLYLQMNSLRD EDTAVYYCARDPLLAGATFFDYWGQGTLVTVSSAstkgpsvfplapcsrstsestaalgclykdyfpepytyswnsgaltsgyhtfpavlqssglyslssvvtypssslgtktytcnvdhkpsntkvdkrveskygppcppcpapeflggpsvflfppkpkdtlmisrtpevtcvvvdvsqedpevqfnwyvdgvevhnaktkpreeqfnstyrvvsyltylhqdwingkeykckvsnkglpssiektiskakgqprepqvytlppsqeemtknqvsltclykgfypsdiavewesngqpennykttppvldsdgsfflysrltvdksrwqegnyfscsvmhealhnhytqkslslspgkSEQ ID NO: 94  8.10.3-CG4 Heavy Chain [Gamma chain] protein sequence melglcwvflvailegvqcEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFSMT WVRQAPGKGLEWVSYISSRSSTISYADSVKGRFTISRDNAKNSLYLQMNSLRD EDTAVYYCARDPLLAGATFFDYWGQGTLVTVSSAstkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtktytcnvdhkpsntkvdkryeskygppcpscpapeflggpsvflfppkpkdtlmisrtpevtcvvvdvsqedpevqfnwyvdgvevhnaktkpreeqfnstyrvvsvltvlhqdwlngkeykckvsnkglpssiektiskakgqprepqvytlppsqeemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflysrltvdksrwqegnvfscsvmhealhnhytqkslslspgkSEQ ID NO: 97  8.10.3FG1 Heavy Chain nucleotide sequence atggagttggggctgagctgggttttccttgttgctattataaaaggtgtccagtgtGAGGTGCAGCTGGTG GAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCC TGTGCAGCCTCTGGATTCACCTTCAGTAGTTTTAGTATGACCTGGGTCC GCCAGGCTCCAGGAAAGGGGCTGGAGTGGGTTTCATACATTAGTAGTA GAAGTAGTACCATATCCTACGCAGACTCTGTGAAGGGCCGATTCACCA TCTCCAGAGACAATGCCAAGAACTCACTGTATCTGCAAATGAACAGCC TGAGAGACGAGGACACGGCTGTGTATTACTGTGCGAGAGATCCTCTTCT AGCGGGAGCTACCTTCTTTGACTACTGGGGCCAGGGAACCCTGGTCAC CGTCTCCTCAGCCtccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctggg ggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccc tgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgc cctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggiggacaagaa agttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagt cttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtg agccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgc gggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaa ggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcag ccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcc tggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagac cacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagca ggggaacgtcnctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgictccg ggtaaatag  SEQ ID NO: 98 8.10.3FG1 Heavy chain (gamma chain) protein sequence melglcwvflvailegvqcEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFSMT WVRQAPGKGLEWVSYISSRSSTISYADSVKGRFTISRDNAKNSLYLQMNSLRD EDTAVYYCARDPLLAGATFFDYWGQGTLVTVSSAstkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsyltvlhqdwingkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk SEQ ID NO: 33  9.7.2IF Heavy Chain [Gamma chain]nucleotide sequence atggagtttgggctgagctgggttttccttgttgctattataaaaggtgtccagtgtcAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTAGTAGTGGTAGTACCATATACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAATTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGGCGTATAGGAGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTtccaccaagggcccatccgtcttccccctggcgccctgctctagaagcacctccgagagcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatga tctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtac gtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtca gcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctccca gcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatccc gggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtgga gtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcc tctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggct ctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa  SEQ ID NO: 34 9.7.2IF Heavy Chain [Gamma Chain] protein sequence mefglswvflvaiikgvqcQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCARRIGGMDVWGQGTTVTVSSAstkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssnfgtqtytcnvdhkpsntkvdktverkccvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyvdgvevhnaktkpreeqfnstfrvvsvltvvhqdwlngkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkSEQ ID NO: 35  9.7.2IF Light Chain [Kappa chain] nucleotide sequence atggacatgagggtccccgctcagctcctggggctcctgctactctggctccgaggtgccagatgtGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCGGCTTTTTAATTTGGTATCAGCAGAGACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTACATCCAGTTTACAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGAactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtgga aggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctaca gcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatca gggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt  SEQ ID NO: 36 9.7.2IF Light Chain [Kappa chain] protein sequence mdmrvpaqllgllllwlrgarcDIQMTQSPSSLSASVGDRVTITCRASQSISGFLI WYQQRPGKAPKLLIYATSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTFGPGTKVDIKRtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgecSEQ ID NO: 45  9.7.2 Heavy Chain [Gamma chain] nucleotide sequence atggagtttgggctgagctgggttttccttgttgctattataaaaggtgtccagtgtcAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTAGTAGTGGTAGTACCATATACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAATTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGGCGTATAGGAGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTtccaccaagggcccatccgtcttccccctggcgccctgctctagaagcacctccgagagcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacgaagac ctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagagagttgagtccaaatatggtcccccat gcccatcatgcccagcacctgagttcctggggggaccatcagtcttcctgttccccccaaaacccaaggacactctcat gatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggt acgtggatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggt cagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctc ccgtcctccatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccat cccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgt ggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttc ttcctctacagcaggctaaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgag gctctgcacaaccactacacacagaagagcctctccctgtctccgggtaaa  SEQ ID NO: 46 9.7.2 Heavy Chain [Gamma Chain] protein sequence mefglswvflvaiikgvqcQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCARRIGGMDVWGQGTTVTVSSAstkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtktytcnvdhkpsntkvdkrveskygppcpscpapeflggpsvflfppkpkdtlmisrtpevtcvvvdvsqedpevqfnwyvdgvevhnaktkpreeqfnstyrvvsvltvlhqdwlngkeykckvsnkglpssiektiskakgqprepqvytlppsqeemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflysrltvdksrwqegnvfscsvmhealhnhytqkslslspgkSEQ ID NO: 47  9.7.2 and 9.7.2-Ser Light Chain [Kappa chain]nucleotide sequence atggacatgagggtccccgctcagctcctggggctcctgctactctggctccgaggtgccagatgtGACATCC AGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGT CACCATCACTTGCCGGGCAAGTCAGAGCATTAGCGGCTTTTTAATTTGG TATCAGCAGAGACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTACA TCCAGTTTACAAAGTGGGGTCCCATTAAGGTTCAGTGGCAGTGAATCTG GGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGC AACTTACTACTGTCAACAGAGTTACAGTACCCCATTCACTTTCGGCCCT GGGACCAAAGTGGATATCAAACGAactgtggctgcaccatctgtcttcatcttcccgccatctga tgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtgga aggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctaca gcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatca gggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt  SEQ ID NO: 48 9.7.2 and 9.7.2-Ser Light Chain [Kappa chain] protein sequence mdmrvpaqllgllllwlrgarcDIQMTQSPSSLSASVGDRVTITCRASQSISGFLI WYQQRPGKAPKLLIYATSSLQSGVPLRFSGSESGTDFTLTISSLQPEDFATYYCQQSYSTPFTFGPGTKVDIKRtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgecSEQ ID NO: 50  9.7.2C-Ser Heavy Chain [Gamma chain] protein sequence mefglswvflvaiikgvqcQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCAIRIGGMDVWGQGTTVTVSSAstkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtktytcnvdhkpsntkvdkryeskygppcppcpapeflggpsvflfppkpkdtlmisrtpevtcvvvdvsqedpevqfnwyvdgvevhnaktkpreeqfnstyrvvsvltvlhqdwlngkeykckvsnkglpssiektiskakgqprepqvytlppsqeemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflysrltvdksrwqegnvfscsvmhealhnhytqkslslspgkSEQ ID NO: 52 9.7.2C-Ser, 9.7.2-CG2 and 9.7.2-CG4 Light Chain [Kappa chain]protein sequence mdmrvpaqllgllllwlrgarcDIQMTQSPSSLSASVGDRVTITCRASQSISGFLI WYQQKPGKAPKLLIYATSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTFGPGTKVDIKRtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgecSEQ ID NO: 66  9.7.2-CG2 Heavy Chain [Gamma chain] protein sequence mefglswvflvaiikgvqcQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCAIRIGGMDVWGQGTTVTVSSAstkgpsvfplapcsrstsestaalgclvkdyfpepytyswnsgaltsgyhtfpavlqssglysissvvtypssnfgtqtytcnvdhkpsntkvdktverkccvecppcpappvagpsvflfppkpkdtlmisrtpevtcvvvdvshedpevqfnwyydgvevhnaktkpreeqfnstfrvvsvltvvhqdwingkeykckvsnkglpapiektisktkgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppmldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkSEQ ID NO: 70  9.7.2-CG4 Heavy Chain [Gamma chain] protein sequence mefglswvflvaiikgvqcQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCARIGGMDVWGQGTTVTVSSAstkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgyhtfpaylqssglysissvvtypssslgtktytcnvdhkpsntkvdkrveskygppcpscpapeflggpsvflfppkpkdtlmisrtpevtcvvvdvsqedpevqfnwyvdgvevhnaktkpreeqfnstvrvvsvltvlhqdwingkeykckvsnkglpssiektiskakgqprepqvytlppsqeemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflysrltydksrwqegnvfscsvmhealhnhytqkslslspgkSEQ ID NO: 86  9.7.2-Ser Heavy Chain [Gamma chain] protein sequence mefglswvflvaiikgvqcQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCARRIGGMDVWGQGTTVTVSSAstkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgyhtfpavlqssglyslssvvtypssslgtktytcnvdhkpsntkvdkrveskygppcppcpapeflggpsvflfppkpkdtlmisrtpevtcvvvdvsqedpevqfnwyvdgvevhnaktkpreeqfnstvrvvsvltvlhqdwingkeykckvsnkglpssiektiskakgqprepqvytlppsqeemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflysrltvdksrwqegnvfscsvmhealhnhytqkslslspgkKey: Signal peptide: underlined lower case CDRs 1,2,3: underlinedUPPERCASE Variable domain: UPPER CASE Constant domain: lower caseMutations from germline in bold

What is claimed:
 1. A monoclonal antibody or an antigen-binding fragmentthereof that specifically binds macrophage colony stimulating factor(M-CSF), wherein the antibody comprises the amino acid sequences of thecomplementarity determining region (CDR) 1, CDR2 and CDR3 in SEQ ID NO:34 and the amino acid sequences of the CDR1, CDR2 and CDR3 in SEQ ID NO:36.
 2. The monoclonal antibody or antigen-binding fragment according toclaim 1, wherein the antibody comprises the heavy chain variable domainin SEQ ID NO: 34 without the signal sequence, and the light chainvariable domain in SEQ ID NO: 36 without the signal sequence.
 3. Themonoclonal antibody or antigen-binding fragment according to claim 2,wherein the heavy chain amino acid sequence of the monoclonal antibodyis SEQ ID NO: 34 without the signal sequence, and the light chain aminoacid sequence of the monoclonal antibody is SEQ ID NO: 36 without thesignal sequence.
 4. The monoclonal antibody or antigen-binding fragmentaccording to claim 1, wherein the antibody is an IgG molecule.
 5. Themonoclonal antibody or antigen-binding fragment according to claim 2,wherein the antibody is an IgG molecule.
 6. The antigen-binding fragmentaccording to claim 1, wherein the fragment is selected from the groupconsisting of: an Fab fragment, an F(ab′)₂ fragment and an Fv fragment.7. The antigen-binding fragment according to claim 2, wherein thefragment is selected from the group consisting of: an Fab fragment, anF(ab′)₂ fragment and an Fv fragment.
 8. A pharmaceutical compositioncomprising the monoclonal antibody according to claim 1 and apharmaceutically acceptable carrier.
 9. The pharmaceutical compositionaccording to claim 8, wherein the antibody comprises the heavy chainvariable domain in SEQ ID NO: 34 without the signal sequence, and thelight chain variable domain in SEQ ID NO: 36 without the signalsequence.
 10. The pharmaceutical composition according to claim 9,wherein the heavy chain amino acid sequence of the monoclonal antibodyis SEQ ID NO: 34 without the signal sequence, and the light chain aminoacid sequence of the monoclonal antibody is SEQ ID NO: 36 without thesignal sequence.
 11. The pharmaceutical composition according to claim8, wherein the antibody is an IgG molecule.
 12. The pharmaceuticalcomposition according to claim 8, wherein the fragment is selected fromthe group consisting of: an Fab fragment, an F(ab′)₂ fragment and an Fvfragment.